Pim kinase inhibitors and methods of their use

ABSTRACT

The present invention relates to new compounds of Formulas I and II, their tautomers, stereoisomers and polymorphs, and pharmaceutically acceptable salts, esters, metabolites or prodrugs thereof, compositions of the new compounds together with pharmaceutically acceptable carriers, and uses of the new compounds, either alone or in combination with at least one additional therapeutic agent, in the inhibition of Pim kinase activity and/or the prophylaxis or treatment of cancer.

FIELD OF THE INVENTION

The present invention relates to new compounds, their tautomers,stereoisomers and polymorphs, and pharmaceutically acceptable salts,esters, metabolites or prodrugs thereof, compositions of the newcompounds together with pharmaceutically acceptable carriers, and usesof the new compounds, either alone or in combination with at least oneadditional therapeutic agent, in the prophylaxis or treatment of cancer.

BACKGROUND

Infection with the Maloney retrovirus and genome integration in the hostcell genome results in development of lymphomas in mice. ProvirusIntegration of Maloney Kinase (PIM-Kinase) was identified as one of thefrequent proto-oncogenes capable of being transcriptionally activated bythis retrovirus integration event (Cuypers H T et al., “Murine leukemiavirus-induced T-cell lymphomagenesis: integration of proviruses in adistinct chromosomal region,” Cell 37(1):141-50 (1984); Selten G, etal., “Proviral activation of the putative oncogene Pim-1 in MuLV inducedT-cell lymphomas” EMBO J 4(7): 1793-8 (1985)), thus establishing acorrelation between over-expression of this kinase and its oncogenicpotential. Sequence homology analysis demonstrated that there are 3highly homologous Pim-Kinases (Pim1, 2 & 3), Pim1 being theproto-oncogene originally identified by retrovirus integration.Furthermore, transgenic mice over-expressing Pim1 or Pim2 show increasedincidence of T-cell lymphomas (Breuer M et al., “Very high frequency oflymphoma induction by a chemical carcinogen in pim-1 transgenic mice”Nature 340(6228):61-3 (1989)), while over-expression in conjunction withc-myc is associated with incidence of B-cell lymphomas (Verbeek S etal., “Mice bearing the E mu-myc and E mu-pim-1 transgenes developpre-B-cell leukemia prenatally” Mol Cell Biol 11(2):1176-9 (1991)).Thus, these animal models establish a strong correlation between Pimover-expression and oncogenesis in hematopoietic malignancies. Inaddition to these animal models, Pim over-expression has been reportedin many other human malignancies. Pim1, 2 & 3 over-expression isfrequently observed in many hematopoietic maligmancies (Amson R et al.,“The human protooncogene product p33pim is expressed during fetalhematopoiesis and in diverse leukemias,” PNAS USA 86(22):8857-61 (1989);Cohen A M et al., “Increased expression of the hPim-2 gene in humanchronic lymphocytic leukemia and non-Hodgkin lymphoma,” Leuk Lymph45(5):951-5 (2004), Huttmann A et al., “Gene expression signaturesseparate B-cell chronic lymphocytic leukaemia prognostic subgroupsdefined by ZAP-70 and CD38 expression status,” Leukemia 20:1774-1782(2006)) and in prostate cancer (Dhanasekaran S M, et al., “Delineationof prognostic biomarkers in prostate cancer,” Nature 412(6849):822-6(2001); Cibull T L, et al., “Overexpression of Pim-1 during progressionof prostatic adenocarcinoma,” J Clin Pathol 59(3):285-8 (2006)), whileover-expression of Pim3 is frequently observed in hepatocellularcarcinoma (Fujii C, et al., “Aberrant expression of serine/threoninekinase Pim-3 in hepatocellular carcinoma development and its role in theproliferation of human hepatoma cell lines,” Int J Cancer 114:209-218(2005)) and pancreatic cancer (Li Y Y et al., “Pim-3, a proto-oncogenewith serine/threonine kinase activity, is aberrantly expressed in humanpancreatic cancer and phosphorylates bad to block bad-mediated apoptosisin human pancreatic cancer cell lines,” Cancer Res 66(13):6741-7(2006)).

Pim1, 2 & 3 are Serine/Threonine kinases normally function in survivaland proliferation of hematopoietic cells in response to growth factorsand cytokines. Cytokines signaling through the Jak/Stat pathway leads toactivation of transcription of the Pim genes and synthesis of theproteins. No further post-translational modifications are required forthe Kinase Pim activity. Thus, signaling down stream is primarilycontrolled at the transcriptional/translational and protein turnoverlevel. Substrates for Pim kinases include regulators of apoptosis suchas the Bcl-2 family member BAD (Aho T et al., “Pim-1 kinase promotesinactivation of the pro-apoptotic Bad protein by phosphorylating it onthe Ser112 gatekeeper site,: FEBS Letters 571: 43-49 (2004)), cell cycleregulators such as p21^(WFA1/CIP1) (Wang Z, et al., “Phosphorylation ofthe cell cycle inhibitor p21Cip1/WAF1 by Pim-1 kinase,” Biochim BiophysActa 1593:45-55 (2002)), CDC25A (1999), C-TAK (Bachmann M et al., “TheOncogenic Serine/Threonine Kinase Pim-1 Phosphorylates and Inhibits theActivity of Cdc25C-associated Kinase 1 (C-TAK1). A novel role for Pim-1at the G2/M cell cycle checkpoint,” J Biol Chem 179:48319-48328 (2004))and NuMA (Bhattacharya N, et al., “Pim-1 associates with proteincomplexes necessary for mitosis, “Chromosoma 111(2):80-95 (2002)) andthe protein synthesis regulator 4EBP1 (Hammerman P S et al, “Pim and Aktoncogenes are independent regulators of hematopoietic cell growth andsurvival,” Blood 105(11):4477-83 (2005)). The effects of Pim(s) in theseregulators are consistent with a role in protection from apoptosis andpromotion of cell proliferation and growth. Thus, over-expression ofPim(s) in cancer is thought to play a role in promoting survival andproliferation of cancer cells and, therefore, their inhibitions shouldbe an effective way of treating cancers on which they areover-expressed. In fact several reports indicate that knocking downexpression of Pim(s) with siRNA results in inhibition of proliferationand cell death (Dai J M, et al., “Antisense oligodeoxynucleotidestargeting the serine/threonine kinase Pim-2 inhibited proliferation ofDU-145 cells,” Acta Pharmacol Sin 26(3):364-8 (2005); Fujii et al 2005;Li et al 2006). Furthermore, mutational activation of several well knowoncogenes in hematopoietic malignancies are thought exert its effects atleast in part through Pim(s). For example, targeted down regulation ofpim expression impairs survival of hematopoietic cells transformed byFlt3 and BCR/ABL (Adam et al 2006). Thus, inhibitors to Pim1, 2 & 3would be useful in the treatment of these malignancies. In addition to apotential role in cancer treatment and myeloproliferative diseases, suchinhibitor could be useful to control expansion of immune cells in otherpathologic condition such as autoimmune diseases, allergic reactions andin organ transplantation rejection syndromes. This notion is supportedby the findings that differentiation of Th1 Helper T-cells by IL-12 andIFN-α results in induction of expression of both Pim1&2 (Aho T et al,“Expression of human Pim family genes is selectively up-regulated bycytokines promoting T helper type 1, but not T helper type 2, celldifferentiation,” Immunology 116: 82-88 (2005)). Moreover, Pim(s)expression is inhibited in both cell types by the immunosuppressiveTGF-β (Aho et al 2005). These results suggest that Pim kinases areinvolved in the early differentiation process of Helper T-cells, whichcoordinate the immunological responses in autoimmune diseases, allergicreaction and tissue transplant rejection.

A continuing need exists for compounds that inhibit the proliferation ofcapillaries, inhibit the growth of tumors, treat cancer, modulate cellcycle arrest, and/or inhibit molecules such as Pim1, Pim2, and Pim3 andpharmaceutical formulations and medicaments that contain such compounds.A need also exists for methods of administering such compounds,pharmaceutical formulations, and medicaments to patients or subjects inneed thereof.

SUMMARY OF THE INVENTION

The present invention provides a compound of Formula I, or astereoisomer, tautomer, or pharmaceutically acceptable salt thereof,

wherein,

R₁ is selected from

X represents CH, or N;

R_(2a) is selected from amino, methyl, CH₂F, CF₃, C₂H₅, and H;

R_(2b) is selected from H, and methyl;

R₃ is selected from H, OH, OCH₃, CH₃, F, and Cl;

R_(4a) is selected from amino, methyl, OH, OCH₃, OC₂H₅, F, CF₃, H, andethyl;

R_(4b) is selected from methyl, H, and F;

R₂₁ represents H or F;

R₂₂ represents H, Cl, or F;

R₂₃ represents F, OC₂H₅, OCH₃, Cl, H, methyl, OH, or OCH(CH₃)₂; and

R₂₄ represents H or OH.

In another aspect, the present invention provides a compound of FormulaII, or a stereoisomer, tautomer, or pharmaceutically acceptable saltthereof,

wherein:

R₁ is selected from —NH—CO-alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl;

X represents CH or N;

R_(2a) is selected from —H, —OH, alkyl, alkoxy, haloalkyl, aminoalkyl,hydroxyalkyl, halo, amino and bezoate;

R_(2b) is selected from —H and alkyl;

R₃ is selected from H, OH, alkyl, alkoxy and halo;

R_(4a) is selected from —OH, alkyl, alkoxy, haloalkyl, aminoalkyl,hydroxyalkyl, halo and amino; and

R_(4b) is selected from H, alkyl and halo.

Another aspect of the present invention provides a compositioncomprising a therapeutically effective amount of compound of Formula Ior Formula II, or a stereoisomer, tautomer, or pharmaceuticallyacceptable salt thereof, together with a pharmaceutically acceptablecarrier.

Provided in another aspect of the present invention is a method forinhibiting PIM kinase activity in a cell, comprising contacting the cellwith an effective amount of a compound of Formula I or Formula II. Yetanother aspect of the present invention provides a method for treating acondition by modulation of Provirus Integration of Maloney Kinase (PIMkinase) activity comprising administering to a patient in need of suchtreatment an effective amount of a compound of Formula I or Formula II.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides compounds of Formula I, ora stereoisomer, tautomer, or pharmaceutically acceptable salt thereof,

wherein,

R₁ is selected from

X represents CH, or N;

R_(2a) is selected from amino, methyl, CH₂F, CF₃, C₂H₅, and H;

R_(2b) is selected from H, and methyl;

R₃ is selected from H, OH, OCH₃, CH₃, F, and Cl;

R_(4a) is selected from amino, methyl, OH, OCH₃, OC₂H₅, F, CF₃, H, andethyl;

R_(4b) is selected from methyl, H, and F;

R₂₁ represents H or F;

R₂₂ represents H, Cl, or F;

R₂₃ represents F, OC₂H₅, OCH₃, Cl, H, methyl, OH, or OCH(CH₃)₂; and

R₂₄ represents H or OH.

In another aspect, the present invention provides a compound of FormulaII, or a stereoisomer, tautomer, or pharmaceutically acceptable saltthereof,

wherein:

R₁ is selected from —NH—CO-alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl;

X represents CH or N;

R_(2a) is selected from —H, —OH, alkyl, alkoxy, haloalkyl, aminoalkyl,hydroxyalkyl, halo, amino and bezoate;

R_(2b) is selected from —H and alkyl;

R₃ is selected from H, OH, alkyl, alkoxy and halo;

R_(4a) is selected from —OH, alkyl; alkoxy, haloalkyl, aminoalkyl,hydroxyalkyl, halo and amino; and

R_(4b) is selected from H, alkyl and halo.

In some aspects, the invention provides compounds of Formula II whereinR₁ is selected from substituted or unsubstituted phenyl, substituted orunsubstituted cyclohexyl, and substituted or unsubstituted piperidinyl.In other aspects, the invention provides compounds of Formula II whereinR₁ is selected from

wherein:

R₂₁ is H or halo;

R₂₂ is H or halo;

R₂₃ is selected from H, halo, alkyl and alkoxy; and

R₂₄ is H or OH.

In some embodiments of the invention, R₂₁ and R₂₂ are independentlyselected from H or F. In other embodiments, R₂₃ is selected from H, Cl,F, —OC₂H₅, —OCH₃, and —OCH(CH₃)₂.

In other embodiments, the invention provides compounds of formula IIwherein R₂ is selected from H, methyl, ethyl, methoxy, ethoxy,fluoromethyl, trifluoromethyl, aminomethyl and hydroxymethyl.

Yet other embodiments provide compounds of formula II wherein R₃ isselected from H, —OH, methyl, methoxy, F and Cl.

In some embodiments, the invention provides compounds of formula IIwherein R_(4a) is selected from —OH, methyl, ethyl, trifluoromethyl,methoxy, ethoxy, amino, F and Cl. Other embodiments provide compounds offormula II wherein R_(4b) is selected from methyl and F.

In some presently preferred embodiments, the invention provides acompound of Formula I or Formula II selected from the group consistingof(S)-5-amino-N-(4-(3-aminopiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-methylpiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methyl-piperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,5-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-(trifluoromethyl)piperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-ethylpiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluoro-4-hydroxyphenyl)thiazole-4-carboxamide,5-amino-N-(4-(3-amino-4-hydroxycyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-(3-amino-5-(fluoromethyl)piperidin-1-yl)-pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluoro-3-methoxyphenyl)thiazole-4-carboxamide,5-amino-2-(2,6-difluorophenyl)-N-(4-((1R,3S,5S)-3-hydroxy-5-methylcyclohexyl)pyridin-3-yl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-(3-amino-4-methylpiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(3-ethoxy-2,6-difluorophenyl)-thiazole-4-carboxamide,or a stereoisomer, tautomer, or pharmaceutically acceptable saltthereof.

In other presently preferred embodiments, the invention provides acompound of Formula I or Formula II selected from the group consistingof(S)-5-amino-N-(4-(3-aminopiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-methylpiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methyl-piperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,5-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-(trifluoromethyl)piperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-ethylpiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluoro-4-hydroxyphenyl)thiazole-4-carboxamide,5-amino-N-(4-(3-amino-4-hydroxycyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-(3-amino-5-(fluoromethyl)piperidin-1-yl)-pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluoro-3-methoxyphenyl)thiazole-4-carboxamide,5-amino-2-(2,6-difluorophenyl)-N-(4-((1R,3S,5S)-3-hydroxy-5-methylcyclohexyl)pyridin-3-yl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,or a stereoisomer, tautomer, or pharmaceutically acceptable saltthereof.

Another aspect of the present invention provides a compositioncomprising a therapeutically effective amount of compound of Formula Ior Formula II, or a stereoisomer, tautomer, or pharmaceuticallyacceptable salt thereof, together with a pharmaceutically acceptablecarrier.

Provided in another aspect of the present invention is a method forinhibiting PIM kinase activity in a cell, comprising contacting the cellwith an effective amount of a compound of Formula I or Formula II. Yetanother aspect of the present invention provides a method for treating acondition by modulation of Provirus Integration of Maloney Kinase (PIMkinase) activity comprising administering to a patient in need of suchtreatment an effective amount of a compound of Formula I or Formula II.

A preferred embodiment of this aspect of the present invention providesa method for treating a cancer disorder in a patient, comprisingadministering to the patient a composition comprising an amount of acompound of claim 1 or claim 10 effective to inhibit PIM kinase activityin the patient

Other aspect of the present invention provides a compound of any FormulaI or Formula II for use as a therapeutic agent. Yet another aspect ofthe present invention provides the use of any one of the compounds ofFormula I or Formula II in the manufacture of a medicament for thetreatment of cancer.

Definitions

“PIM inhibitor” is used herein to refer to a compound that exhibits anIC₅₀ with respect to PIM Kinase activity of no more than about 100 μMand more typically not more than about 50 μM, as measured in the PIMdepletion assays described herein below.

The phrase “alkyl” refers to alkyl groups that do not containheteroatoms. Thus the phrase includes straight chain alkyl groups suchas methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl and the like.

As used herein, the term “halogen” or “halo” refers to chloro, bromo,fluoro and iodo groups. “Haloalkyl” refers to an alkyl radicalsubstituted with one or more halogen atoms. The term “haloloweralkyl”refers to a lower alkyl radical substituted with one or more halogenatoms. The term “haloalkoxy” refers to an alkoxy radical substitutedwith one or more halogen atoms. The term “haloloweralkoxy” refers to aloweralkoxy radical substituted with one or more halogen atoms.

“Amino” refers herein to the group —NH₂, which may be substituted toform —NRR′. The term “alkylamino” refers herein to the group —NRR′ whereR and R′ are each independently selected from hydrogen or a lower alkyl.The term “arylamino” refers herein to the group —NRR′ where R is aryland R′ is hydrogen, a lower alkyl, or an aryl. The term “aralkylamino”refers herein to the group —NRR′ where R is a lower aralkyl and R′ ishydrogen, a loweralkyl, an aryl, or a loweraralkyl.

The term “alkoxy” refers to RO— wherein R is substituted orunsubstituted alkyl. Representative examples of loweralkoxy groupsinclude methoxy, ethoxy, t-butoxy, trifluoromethoxy and the like.

“Cycloalkyl” refers to a mono- or polycyclic, heterocyclic orcarbocyclic alkyl substituent. Typical cycloalkyl substituents have from3 to 8 backbone (i.e., ring) atoms in which each backbone atom is eithercarbon or a heteroatom. The term “heterocycloalkyl” or “heterocyclyl”refers herein to cycloalkyl substituents that have from 1 to 5, and moretypically from 1 to 4 heteroatoms in the ring structure. Suitableheteroatoms employed in compounds of the present invention are nitrogen,oxygen, and sulfur. Representative heterocycloalkyl moieties include,for example, morpholino, piperazinyl, piperidinyl and the like.Carbocycloalkyl groups are cycloalkyl groups in which all ring atoms arecarbon. When used in connection with cycloalkyl substituents, the term“polycyclic” refers herein to fused and non-fused alkyl cyclicstructures.

“Aryl” refers to optionally substituted monocyclic and polycyclicaromatic groups having from 3 to 14 backbone carbon or hetero atoms, andincludes both carbocyclic aryl groups and heterocyclic aryl groups.Carbocyclic aryl groups are aryl groups in which all ring atoms in thearomatic ring are carbon. The term “heteroaryl” refers herein to arylgroups having from 1 to 4 heteroatoms as ring atoms in an aromatic ringwith the remainder of the ring atoms being carbon atoms. When used inconnection with aryl substituents, the term “polycyclic aryl” refersherein to fused and non-fused cyclic structures in which at least onecyclic structure is aromatic, such as, for example, benzodioxozolo(which has a heterocyclic structure fused to a phenyl group, i.e.,

naphthyl, and the like. Exemplary aryl moieties employed as substituentsin compounds of the present invention include phenyl, pyridyl,pyrimidinyl, thiazolyl, indolyl, imidazolyl, oxadiazolyl, tetrazolyl,pyrazinyl, triazolyl, thiophenyl, furanyl, quinolinyl, purinyl,naphthyl, benzothiazolyl, benzopyridyl, and benzimidazolyl, and thelike.

“Optionally substituted” or “substituted” refers to the replacement ofone or more hydrogen atoms with a monovalent or divalent radical.Suitable substitution groups include, for example, hydroxy, nitro,amino, imino, cyano, halo, thio, sulfonyl, thioamido, amidino, imidino,oxo, oxamidino, methoxamidino, imidino, guanidino, sulfonamido,carboxyl, formyl, loweralkyl, haloloweralkyl, loweralkylamino,haloloweralkylamino, loweralkoxy, haloloweralkoxy, loweralkoxyalkyl,alkylcarbonyl, aminocarbonyl, arylcarbonyl, aralkylcarbonyl,heteroarylcarbonyl, heteroaralkylcarbonyl, alkylthio, aminoalkyl,cyanoalkyl, aryl and the like.

The substitution group can itself be substituted. The group substitutedonto the substitution group can be carboxyl, halo; nitro, amino, cyano,hydroxy, loweralkyl, loweralkoxy, aminocarbonyl, —SR, thioamido, —SO₃H,—SO₂R or cycloalkyl, where R is typically hydrogen, hydroxyl orloweralkyl.

It is understood that the above definitions are not intended to includeimpermissible substitution patterns (e.g., methyl substituted with fivefluoro groups or a halogen atom substituted with another halogen atom).Such impermissible substitution patterns are well known to the skilledartisan.

It will also be apparent to those skilled in the art that the compoundsof the invention, including the compounds of compounds of formulas (I)or (II) or their stereoisomers, as well as the pharmaceuticallyacceptable salts, esters, metabolites and prodrugs of any of them, maybe subject to tautomerization and may therefore exist in varioustautomeric forms wherein a proton of one atom of a molecule shifts toanother atom and the chemical bonds between the atoms of the moleculesare consequently rearranged. See, e.g., March, Advanced OrganicChemistry: Reactions, Mechanisms and Structures, Fourth Edition, JohnWiley & Sons, pages 69-74 (1992). As used herein, the term “tautomer”refers to the compounds produced by the proton shift, and it should beunderstood that the all tautomeric forms, insofar as they may exist, areincluded within the invention.

The compounds of the invention, including the compounds of formulas (I)or (II) or their tautomers, as well as the pharmaceutically acceptablesalts, esters, metabolites and prodrugs of any of them, may compriseasymmetrically substituted carbon atoms. Such asymmetrically substitutedcarbon atoms can result in the compounds of the invention existing inenantiomers, diastereomers, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, such as in (R)— or (S)—forms. As a result, all such possible isomers, individual stereoisomersin their optically pure forms, mixtures thereof, racemic mixtures (or“racemates”), mixtures of diastereomers, as well as single diastereomersof the compounds of the invention are included in the present invention.The terms “S” and “R” configuration, as used herein, are as defined bythe IUPAC 1974 RECOMMENDATIONS FOR SECTION E, FUNDAMENTALSTEREOCHEMISTRY, Pure Appl. Chem. 45:13-30 (1976). The terms α and β areemployed for ring positions of cyclic compounds. The α-side of thereference plane is that side on which the preferred substituent lies atthe lower numbered position. Those substituents lying on the oppositeside of the reference plane are assigned β descriptor. It should benoted that this usage differs from that for cyclic stereoparents, inwhich “α” means “below the plane” and denotes absolute configuration.The terms α and β configuration, as used herein, are as defined by theCHEMICAL ABSTRACTS INDEX GUIDE-APPENDIX IV (1987) paragraph 203.

As used herein, the term “pharmaceutically acceptable salts” refers tothe nontoxic acid or alkaline earth metal salts of the compounds ofFormulas (I) or (II). These salts can be prepared in situ during thefinal isolation and purification of the compounds of Formulas (I) or(II), or by separately reacting the base or acid functions with asuitable organic or inorganic acid or base, respectively. Representativesalts include but are not limited to the following: acetate, adipate,alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate,butyrate, camphorate, camphorsulfonate, digluconate,cyclopentanepropionate, dodecylsulfate, ethanesulfonate,glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate,persulfate, 3-phenylproionate, picrate, pivalate, propionate, succinate,sulfate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate.Also, the basic nitrogen-containing groups can be quaternized with suchagents as loweralkyl halides, such as methyl, ethyl, propyl, and butylchloride, bromides, and iodides; dialkyl sulfates like dimethyl,diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkylhalides like benzyl and phenethyl bromides, and others. Water oroil-soluble or dispersible products are thereby obtained.

Examples of acids which may be employed to form pharmaceuticallyacceptable acid addition salts include such inorganic acids ashydrochloric acid, sulfuric acid and phosphoric acid and such organicacids as oxalic acid, maleic acid, methanesulfonic acid, succinic acidand citric acid. Basic addition salts can be prepared in situ during thefinal isolation and purification of the compounds of formula (I), orseparately by reacting carboxylic acid moieties with a suitable basesuch as the hydroxide, carbonate or bicarbonate of a pharmaceuticallyacceptable metal cation or with ammonia, or an organic primary,secondary or tertiary amine. Pharmaceutically acceptable salts include,but are not limited to, cations based on the alkali and alkaline earthmetals, such as sodium, lithium, potassium, calcium, magnesium, aluminumsalts and the like, as well as nontoxic ammonium, quaternary ammonium,and amine cations, including, but not limited to ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, ethylamine, and the like. Otherrepresentative organic amines useful for the formation of base additionsalts include diethylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine and the like.

As used herein, the term “pharmaceutically acceptable ester” refers toesters, which hydrolyze in vivo and include those that break downreadily in the human body to leave the parent compound or a saltthereof. Suitable ester groups include, for example, those derived frompharmaceutically acceptable aliphatic carboxylic acids, particularlyalkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which eachalkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.Examples of particular esters include formates, acetates, propionates,butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use, aswell as the zwitterionic forms, where possible, of the compounds of theinvention. The term “prodrug” refers to compounds that are rapidlytransformed in vivo to yield the parent compound of the above formula,for example by hydrolysis in blood. A thorough discussion is provided inT. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14of the A.C.S. Symposium Series, and in Edward B. Roche, ed.,Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press, 1987, both of which are incorporatedherein by reference.

It will be apparent to those skilled in the art that the compounds ofthe invention, including the compounds of formulas (I) or (II) or theirtautomers, prodrugs and stereoisomers, as well as the pharmaceuticallyacceptable salts, esters and prodrugs of any of them, may be processedin vivo through metabolism in a human or animal body or cell to producemetabolites. The term “metabolite” as used herein refers to the formulaof any derivative produced in a subject after administration of a parentcompound. The derivatives may be produced from the parent compound byvarious biochemical transformations in the subject such as, for example,oxidation, reduction, hydrolysis, or conjugation and include, forexample, oxides and demethylated derivatives. The metabolites of acompound of the invention may be identified using routine techniquesknown in the art. See, e.g., Bertolini, G. et al., J. Med. Chem.40:2011-2016 (1997); Shan, D. et al., J. Pharm. Sci. 86(7):765-767;Bagshawe K., Drug Dev. Res. 34:220-230 (1995); Bodor, N., Advances inDrug Res. 13:224-331 (1984); Bundgaard, H., Design of Prodrugs (ElsevierPress 1985); and Larsen, I. K., Design and Application of Prodrugs, DrugDesign and Development (Krogsgaard-Larsen et al., eds., Harwood AcademicPublishers, 1991). It should be understood that individual chemicalcompounds that are metabolites of the compounds of formulas (I) or (II)or their tautomers, prodrugs and stereoisomers, as well as thepharmaceutically acceptable salts, esters and prodrugs of any of them,are included within the invention.

The term “cancer” refers to cancer diseases that can be beneficiallytreated by the inhibition of Pim kinase, including, for example, solidcancers, such as carcinomas (e.g., of the lungs, pancreas, thyroid,ovarian, bladder, breast, prostate, or colon), melanomas, myeloiddisorders (e.g., myeloid leukemia, multiple myeloma anderythroleukemia), adenomas (e.g., villous colon adenoma) and sarcomas(e.g., osteosarcoma).

Synthetic Methods

The compounds of the invention can be obtained through procedures knownto the skilled in the art. For example, as shown in Scheme 1, 4-chloro,3-nitro pyridine can be reacted with a nucleophile yielding after nitroreduction a 4-substituted 3-amino pyridine I. The substituted aminopyridines I can be acylated with thiazolecarboxylic acids with the aidof coupling agents, or with acid halides or acid anhydrides yielding 3,4 disubstituted pyridines II. If the 2 position R group of the thiazoleis bromo, triflate or iodo, further modification to incorporate avariety of substituents at this position can be realized by metalmediated carbon-carbon bond forming reactions.

The reaction of 4-chloro-3-nitropyridine with nucleophiles as depictedin Scheme 1 is not limited to nitrogen based nucleophiles; carbon-carbonbonds can be formed as well with the net addition of carbonnucleophiles. As shown in Scheme 2, cyclohexanediones can be convertedvia monotriflates to the corresponding cyclohexenoneboronate esterswhich can undergo palladium mediated carbon-carbon bond formation with4-chloro, 3-nitro pyridine to yield nitropyridine substitutedcyclohexenones III. Reduction of the enone functionality can yield acyclohexenol IV which upon alcohol protection, nitro and alkenereduction, amide coupling and deprotection can yield cyclohexanol amidesV. Cyclohexenol IV can also undergo Mitsunobo reaction with phthalimideto yield a protected aminocyclohexene V. Following nitro and alkenereduction, phthalimide protected aminocyclohexyl pyridyl aniline VIIacan undergo amide coupling and deprotection, to yield aminocyclohexaneamides VIII. The corresponding Boc protected aminocyclohexane pyridylaniline VIIb can also be prepared from cyclohexenol IV in the followingmanner: alcohol protection, alkene and nitro reduction, pyridyl amineCbz protection, silyl ether deprotection, dess-martin oxidation to thecyclohexanone, reductive amination with benzylamine, Cbz and benzyldeprotection and primary aliphatic amine Boc protection. In the amideproducts V and VIII, if R2 is halo or triflate, the amides IV and VIIIcan be further modified by standard modifications to introducesubstituted aryls, alkyls and heteroaryls at R2. For example, if R2 isBr, by reaction with boronic acids or organometallic reagents, orconversion to the corresponding boronate ester and reaction witharyl/heteroaryl halides or triflates, a variety of R2 modifications arepossible.

Thiazole amides with substituted cyclohexyl groups can be obtained bymodification of nitropyridyl cyclohexenol IV. As shown in Scheme 3,cyclohexenol IV can be dehydrated yielding a cyclohexadiene which uponepoxidation (via bromohydrin formation and HBr elimination or from mCPBAdirectly) and azide epoxide opening yields cyclohexenyl azido alcoholIX. Cyclohexenyl azido alcohol IX can be converted to the transprotected amino hydroxy aniline Xa by azide reduction, alcoholprotection and alkene and nitro reduction. Alternatively, thecyclohexenyl azido alcohol IX can be converted to the protected cisamino hydroxy aniline Xb by azide reduction and Boc protection, alcoholmesylation and intramolecular cyclization to the cis cyclic carbamate,followed by Boc protection and alkene and nitro reduction. The resultingcyclohexylpyridyl anilines Xa and Xb can be converted to thecorresponding thiazole amides XIa and XIb by amide coupling, acetate orcyclic carbamate cleavage and Boc deprotection. If R₂ is halo ortriflate, the amides XIa and XIb and XII can be further modified bystandard modifications to introduce substituted aryls, alkyls andheteroaryls at R₂ after amide bond formation and prior to fulldeprotection. For example, if R₂ is Br, by reaction with boronic acidsor organometallic reagents, or conversion to the corresponding boronateester and reaction with aryl/heteroaryl halides or triflates, a varietyof R₂ modifications are possible.

Substituted 3-aminopiperidines can be prepared and modified to yieldsubstituted 3-aminopiperidinyl thiazole amides XII as depicted in Scheme4. Reaction of crotyl boronate esters with SerOBn aldehyde followed bycyclic carbamate formation, alkene oxidative cleavage and reductionyields hydroxyl compound XIII. Benzyl deprotection followed bybistosylation and reaction with p-methoxybenzylamine, and aminedeprotection yields piperidine XIV. By use of chiral boronate esters,and different L and D serine derived aldehydes, all possiblediastereomers of the resulting trisubstituted 5-alkyl, 4-hydroxy,3-aminopiperidine can be obtained. Reaction of substituted piperidineXIV with 4-chloro-3-nitropyridine, followed by carbamate protection,nitro reduction, amide coupling, cyclic carbamate opening anddeprotection yields trisubstituted 5-methyl, 4-hydroxy,3-aminopiperidinyl thiazole amides XII. If R₂ is halo or triflate, theamide XII can be further modified by standard modifications to introducesubstituted aryls, alkyls and heteroaryls at R₂ after amide bondformation and prior to full deprotection. For example, if R₂ is Br, byreaction with boronic acids or organometallic reagents, or conversion tothe corresponding boronate ester and reaction with aryl/heteroarylhalides or triflates, a variety of R2 modifications are possible.

The compounds of the invention are useful in vitro or in vivo ininhibiting the growth of cancer cells. The compounds may be used aloneor in compositions together with a pharmaceutically acceptable carrieror excipient. Suitable pharmaceutically acceptable carriers orexcipients include, for example, processing agents and drug deliverymodifiers and enhancers, such as, for example, calcium phosphate,magnesium stearate, talc, monosaccharides, disaccharides, starch,gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose,dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidinone, lowmelting waxes, ion exchange resins, and the like, as well ascombinations of any two or more thereof. Other suitable pharmaceuticallyacceptable excipients are described in “Remington's PharmaceuticalSciences,” Mack Pub. Co., New Jersey (1991), incorporated herein byreference.

Effective amounts of the compounds of the invention generally includeany amount sufficient to detectably inhibit Pim activity by any of theassays described herein, by other Pim kinase activity assays known tothose having ordinary skill in the art or by detecting an inhibition oralleviation of symptoms of cancer.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. It will beunderstood, however, that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination, and the severity of the particular diseaseundergoing therapy. The therapeutically effective amount for a givensituation can be readily determined by routine experimentation and iswithin the skill and judgment of the ordinary clinician.

For purposes of the present invention, a therapeutically effective dosewill generally be a total daily dose administered to a host in single ordivided doses may be in amounts, for example, of from 0.001 to 1000mg/kg body weight daily and more preferred from 1.0 to 30 mg/kg bodyweight daily. Dosage unit compositions may contain such amounts ofsubmultiples thereof to make up the daily dose.

The compounds of the present invention may be administered orally,parenterally, sublingually, by aerosolization or inhalation spray,rectally, or topically in dosage unit formulations containingconventional nontoxic pharmaceutically acceptable carriers, adjuvants,and vehicles as desired. Topical administration may also involve the useof transdermal administration such as transdermal patches orionophoresis devices. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection, or infusion techniques.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-propanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable nonirritating excipient such as cocoabutter and polyethylene glycols, which are solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum and release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose lactose or starch. Such dosage forms may also comprise, as isnormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, cyclodextrins, and sweetening,flavoring, and perfuming agents.

The compounds of the present invention can also be administered in theform of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multi-lamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto a compound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andphosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art. See, for example, Prescott, Ed.,Methods in Cell Biology, Volume XIV, Academic Press, New York, N.W., p.33 et seq. (1976).

While the compounds of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more other agents used in the treatment of cancer. The compoundsof the present invention are also useful in combination with knowntherapeutic agents and anti-cancer agents, and combinations of thepresently disclosed compounds with other anti-cancer or chemotherapeuticagents are within the scope of the invention. Examples of such agentscan be found in Cancer Principles and Practice of Oncology, V. T. Devitaand S. Hellman (editors), 6^(th) edition (Feb. 15, 2001), LippincottWilliams & Wilkins Publishers. A person of ordinary skill in the artwould be able to discern which combinations of agents would be usefulbased on the particular characteristics of the drugs and the cancerinvolved. Such anti-cancer agents include, but are not limited to, thefollowing: estrogen receptor modulators, androgen receptor modulators,retinoid receptor modulators, cytotoxic/cytostatic agents,antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoAreductase inhibitors and other angiogenesis inhibitors, inhibitors ofcell proliferation and survival signaling, apoptosis inducing agents andagents that interfere with cell cycle checkpoints. The compounds of theinvention are also useful when co-administered with radiation therapy.

Therefore, in one embodiment of the invention, the compounds of theinvention are also used in combination with known anticancer agentsincluding, for example, estrogen receptor modulators, androgen receptormodulators, retinoid receptor modulators, cytotoxic agents,antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoAreductase inhibitors, HIV protease inhibitors, reverse transcriptaseinhibitors, and other angiogenesis inhibitors.

In certain presently preferred embodiments of the invention,representative agents useful in combination with the compounds of theinvention for the treatment of cancer include, for example, irinotecan,topotecan, gemcitabine, 5-fluorouracil, leucovorin carboplatin,cisplatin, taxanes, tezacitabine, cyclophosphamide, vinca alkaloids,imatinib (Gleevec), anthracyclines, rituximab, trastuzumab, as well asother cancer chemotherapeutic agents.

The above compounds to be employed in combination with the compounds ofthe invention will be used in therapeutic amounts as indicated in thePhysicians' Desk Reference (PDR) 47th Edition (1993), which isincorporated herein by reference, or such therapeutically useful amountsas would be known to one of ordinary skill in the art.

The compounds of the invention and the other anticancer agents can beadministered at the recommended maximum clinical dosage or at lowerdoses. Dosage levels of the active compounds in the compositions of theinvention may be varied so as to obtain a desired therapeutic responsedepending on the route of administration, severity of the disease andthe response of the patient. The combination can be administered asseparate compositions or as a single dosage form containing both agents.When administered as a combination, the therapeutic agents can beformulated as separate compositions, which are given at the same time ordifferent times, or the therapeutic agents, can be given as a singlecomposition.

The present invention will be understood more readily by reference tothe following examples, which are provided by way of illustration andare not intended to be limiting of the present invention.

Representative side chains for use in the compounds of the followingexamples may generally be prepared in accordance with the followingprocedures:

EXAMPLES

Referring to the examples that follow, compounds of the preferredembodiments were synthesized using the methods described herein, orother methods, which are known in the art.

The compounds and/or intermediates were characterized by highperformance liquid chromatography (HPLC) using a Waters Milleniumchromatography system with a 2695 Separation Module (Milford, Mass.).The analytical columns were reversed phase Phenomenex Luna C18-5μ,4.6×50 mm, from Alltech (Deerfield, Ill.). A gradient elution was used(flow 2.5 mL/min), typically starting with 5% acetonitrile/95% water andprogressing to 100% acetonitrile over a period of 10 minutes. Allsolvents contained 0.1% trifluoroacetic acid (TFA). Compounds weredetected by ultraviolet light (UV) absorption at either 220 or 254 nm.HPLC solvents were from Burdick and Jackson (Muskegan, Mich.), or FisherScientific (Pittsburgh, Pa.).

In some instances, purity was assessed by thin layer chromatography(TLC) using glass or plastic backed silica gel plates, such as, forexample, Baker-Flex Silica Gel 1B2-F flexible sheets. TLC results werereadily detected visually under ultraviolet light, or by employingwell-known iodine vapor and other various staining techniques.

Mass spectrometric analysis was performed on one of three LCMSinstruments: a Waters System (Alliance HT HPLC and a Micromass ZQ massspectrometer; Column: Eclipse XDB-C18, 2.1×50 mm; gradient: 5-95% (or35-95%, or 65-95% or 95-95%) acetonitrile in water with 0.05% TFA over a4 min period; flow rate 0.8 mL/min; molecular weight range 200-1500;cone Voltage 20 V; column temperature 40° C.), another Waters System(ACQUITY UPLC system and a ZQ 2000 system; Column: ACQUITY UPLC HSS-C18,1.8 um, 2.1×50 mm; gradient: 5-95% (or 35-95%, or 65-95% or 95-95%)acetonitrile in water with 0.05% TFA over a 1.3 min period; flow rate1.2 mL/min; molecular weight range 150-850; cone Voltage 20 V; columntemperature 50° C.) or a Hewlett Packard System (Series 1100 HPLC;Column: Eclipse XDB-C18, 2.1×50 mm; gradient: 5-95% acetonitrile inwater with 0.05% TFA over a 4 min period; flow rate 0.8 mL/min;molecular weight range 150-850; cone Voltage 50 V; column temperature30° C.). All masses were reported as those of the protonated parentions.

GCMS analysis was performed on a Hewlett Packard instrument (HP6890Series gas chromatograph with a Mass Selective Detector 5973; injectorvolume: 1 μL; initial column temperature: 50° C.; final columntemperature: 250° C.; ramp time: 20 minutes; gas flow rate: 1 mL/min;column: 5% phenyl methyl siloxane, Model No. HP 190915-443, dimensions:30.0 m×25 m×0.25 m).

Nuclear magnetic resonance (NMR) analysis can be performed with a Varian300 or 400 MHz NMR (Palo Alto, Calif.). The spectral reference caneither be TMS or the known chemical shift of the solvent.

The purity of some of the compounds is assessed by elemental analysis(Desert Analytics, Tucson, Ariz.).

Melting points are determined on a Laboratory Devices Mel-Temp apparatus(Holliston, Mass.).

Preparative separations are carried out using a Flash 40 chromatographysystem and KP-Sil, 60A (Biotage, Charlottesville, Va.), or by flashcolumn chromatography using silica gel (230-400 mesh) packing material,or by HPLC using a Waters 2767 Sample Manager, C-18 reversed phasecolumn, 30×50 mm, flow 75 mL/min. Typical solvents employed for theFlash 40 Biotage system and flash column chromatography aredichloromethane, methanol, ethyl acetate, hexane, acetone, aqueousammonia (or ammonium hydroxide), and triethyl amine. Typical solventsemployed for the reverse phase HPLC are varying concentrations ofacetonitrile and water with 0.1% trifluoroacetic acid.

It should be understood that the organic compounds according to thepreferred embodiments may exhibit the phenomenon of tautomerism. As thechemical structures within this specification can only represent one ofthe possible tautomeric forms, it should be understood that thepreferred embodiments encompasses any tautomeric form of the drawnstructure.

It is understood that the invention is not limited to the embodimentsset forth herein for illustration, but embraces all such forms thereofas come within the scope of the above disclosure.

The examples below as well as throughout the application, the followingabbreviations have the following meanings. If not defined, the termshave their generally accepted meanings.

Abbreviations

Boc₂O di-tert-butyl dicarbonate DAST (Diethylamino)sulfurtrifluoride DCMDichloromethane DIEA diisopropylethylamine DtBAD Di-tert-butylazodicarboxylate DMA dimethylacetamide DMAP 4-dimethylaminopyridine DME1,2-dimethoxyethane DMF N,N-dimethylformamide DPPF1,1′-bis(diphenylphosphino)ferrocene EDC1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EtOAc ethylacetate EtOH Ethanol HOAT Hydroxyazabenzotriazole MeCN acetonitrile MeOHmethanol Na₂CO₃ sodium carbonate NaHCO₃ sodium bicarbonate NBSN-bromosuccinimide NMP N-methyl-2-pyrrolidone Pd₂(dba)₃Tris(dibenzylideneacetone)dipalladium(0) Pd(PPh₃)₄Tetrakis(triphenylphospine)palladium(0) Pd(dppf)Cl₂-Dichloro-(1,2-bis(diphenylphosphino)ethan)- DCMPalladium(II)-dichloromothethane adduct RT or rt room temperature TDMSClTert-butyldimethylsilylchloride TEA triethylamine THF tetrahydrofuranTf2O Triflic anhydride

Method 1 Synthesis of 3-nitro-4-(piperidin-1-yl)pyridine

A solution of 4-chloro-3-nitropyridine (1.0 equiv.) and piperidine (2.0equiv.) in ethanol, at a concentration of 0.5 M, was stirred at rt for48 hours at which time the ethanol was removed in vacuo. The residue waspartitioned between EtOAc (300 mL) and Na₂CO₃ (sat.) (75 mL), was washedfurther with H₂O (50 mL), NaCl_((sat.)) (50 mL), was dried over MgSO₄,was filtered and the volatiles were removed in vacuo yielding3-nitro-4-(piperidin-1-yl)pyridine (95%). LCMS (m/z): 207.7 (MH⁺); LCR_(t)=1.60 min. ¹H NMR (CDCl₃): δ 8.80 (s, 1H), 8.31 (d, J=5.7, 1H),6.84 (d, J=6.3, 1H), 3.18-3.21 (m, 4H), 1.64-1.78 (m, 6H).

Synthesis of (S)-tert-butyl1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

Method 1 was followed using 1 eq each of 4-chloro-3-nitropyridine,(S)-3-N-Boc-amino piperidine and diisopropylethylamine yielding(S)-tert-butyl 1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate, (99%).LCMS (m/z): 323.1 (MH⁺); LC R_(t)=2.13 min.

Synthesis of trans(±)-Benzyl3-(tert-butoxycarbonylamino)-4-hydroxypiperidine-1-carboxylate

Synthesis of trans(±)-Benzyl4-(tert-butoxycarbonylamino)-3-hydroxypiperidine-1-carboxylate

A solution of (±)benzyl 7-oxa-3-azabicyclo[4.1.0]heptane-3-carboxylate(1.0 equiv.) in saturated ammonium hydroxide aqueous solution andethanol (1:1, 0.05 M solution) in a sealed steel bomb was heated to 70°C. for 5 h. After all volatile materials were removed by N₂ gas stream,ethyl acetate and water were added for work-up. The crude regioisomericmixture, benzyl 3-amino-4-hydroxypiperidine-1-carboxylate and benzyl4-amino-3-hydroxypiperidine-1-carboxylate was reacted with Boc₂O (1.0equiv.) and triethylamine (1.0 equiv.) in dichloromethane (0.1 Msolution). After stirred for 2 h at room temperature, the reactionmixture was extracted with dichloromethane. The polar (±)-benzyl3-(tert-butoxycarbonylamino)-4-hydroxypiperidine-1-carboxylate andnonpolar (±)-benzyl4-(tert-butoxycarbonylamino)-3-hydroxypiperidine-1-carboxylate wereobtained by flash column chromatography (20% to 40% EtOAc in hexanes,28%, 51% each). LCMS (m/z): 351.1 (MH⁺), R_(t)=0.81 min, LCMS (m/z):351.1 (MH⁺), R_(t)=0.83 min. The enantiomerically pure (3S,4S)-benzyl3-(tert-butoxycarbonylamino)-4-hydroxypiperidine-1-carboxylate and(3R,4R)-benzyl3-(tert-butoxycarbonylamino)-4-hydroxypiperidine-1-carboxylate wereresolved by chiral HPLC (For analysis R_(t)=6.8 min and 9.1 minrespectively; n-heptane:ethanol=70:30 (v:v), Chiralpak AD-H prep 250×4.6mm at 1 mL/min. For preparative separation, n-heptane:ethanol=80:20(v:v), Chiralpak AS 50×500 mm. at 90 mL/min).

Method 2 Synthesis of 4-(piperidin-1-yl)pyridin-3-amine

To a solution of 3-nitro-4-(piperidin-1-yl)pyridine (1.0 equiv.) inethanol, at a concentration of 0.1 M, was added 10% palladium on carbon(0.1 eq.). The resultant heterogeneous solution was put under anatmosphere of hydrogen and was stirred for 15 hours. At this time themixture was filtered through a pad of celite eluting with methanol. Thevolatiles were removed in vacuo yielding4-(piperidin-1-yl)pyridin-3-amine (93%) as an oil. LCMS (m/z): 178.0(MH⁺); LC R_(t)=1.68 min. ¹H NMR (CDCl₃): δ 8.01 (s, 1H), 7.96 (d,J=5.4, 1H), 6.78 (d, J=5.1, 1H), 3.64-3.74 (m, 2H), 2.86-2.94 (m, 4H),1.66-1.78 (m, 4H), 1.58-1.64 (m, 2H).

Synthesis of (S)-tert-butyl1-(3-aminopyridin-4-yl)piperidin-3-ylcarbamate

Following Method 2, (S)-tert-butyl1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate was reduced yielding(S)-tert-butyl 1-(3-aminopyridin-4-yl)piperidin-3-ylcarbamate, (78%).LCMS (m/z): 293.1 (MH⁺); LC R_(t)=2.08 min.

Synthesis of (3R,4R)-benzyl3-(tert-butoxycarbonylamino)-4-(tert-butyldimethylsilyloxy)piperidine-1-carboxylate

To a solution of (3R,4R)-benzyl3-(tert-butoxycarbonylamino)-4-hydroxypiperidine-1-carboxylate (1.0equiv.) in dichloromethane (0.1 M solution) was added imidazole (1.1equiv.), DMAP (0.1 equiv.), and TBDMSCl (1.1 equiv.) sequentially. Thereaction mixture was stirred at room temperature for 20 h. After workedup with dichloromethane, the crude material was purified by silicacolumn chromatography (10% to 20% EtOAc in hexanes) yielding(3R,4R)-benzyl3-(tert-butoxycarbonylamino)-4-(tert-butyldimethylsilyloxy)piperidine-1-carboxylate(76%). LCMS (m/z): 365.2 [(M-Boc)H⁺]; LC R_(t)=6.05 min.

Synthesis of (3S,4S)-benzyl3-(tert-butoxycarbonylamino)-4-(tert-butyldimethylsilyloxy)piperidine-1-carboxylate

To a solution of (3S,SR)-benzyl3-(tert-butoxycarbonylamino)-4-hydroxypiperidine-1-carboxylate (1.0equiv.) in dichloromethane (0.1 M solution) was added imidazole (1.1equiv.), DMAP (0.1 equiv.), and TBDMSCl (1.1 equiv.) sequentially Thereaction mixture was stirred at room temperature for 20 h. After workedup with dichloromethane, the crude material was purified by silicacolumn chromatography (10% to 20% EtOAc in hexanes) yielding(3S,4S)-benzyl3-(tert-butoxycarbonylamino)-4-(tert-butyldimethylsilyloxy)piperidine-1-carboxylate.LCMS (m/z): 365.2 [(M-Boc)H⁺]; LC R_(t)=6.05 min.

Synthesis of (3R,4R)-Benzyl3-(tert-butoxycarbonylamino)-4-fluoropiperidine-1-carboxylate and(3S,4S)-Benzyl3-(tert-butoxycarbonylamino)-4-fluoropiperidine-1-carboxylate

To a solution of (±)-benzyl3-(tert-butoxycarbonylamino)-4-hydroxypiperidine-1-carboxylate (1.0equiv.) in dichloromethane (0.3 M solution) was added DAST at −78° C.The reaction mixture was slowly warmed up to room temperature for 15 h.After quenched with saturated sodium bicarbonate aqueous solution, ethylacetate and water were added for work-up. The (±)-benzyl3-(tert-butoxycarbonylamino)-4-fluoropiperidine-1-carboxylate wasobtained by silica column chromatography (30% EtOAc in hexanes, 40%).LCMS (m/z): 253.1[(M-Boc)H⁺]; LC R_(t)=4.08 min. The enantiomericallypure (3R,4R)-benzyl3-(tert-butoxycarbonylamino)-4-fluoropiperidine-1-carboxy late and(3S,4S)-benzyl3-(tert-butoxycarbonylamino)-4-fluoropiperidine-1-carboxylate wereresolved by chiral HPLC (for analysis: R_(t)=9.4 min and 12.6 minrespectively; n-heptane:isopropanol=90:10 (v:v), Chiralpak AS 250×4.6 mmat 1 mL/min. For preparative separation, n-heptane:isopropanol=90:10(v:v), Chiralpak AS 50×500 mm. at 90 mL/min).

Synthesis of tert-butyl(3R,4R)-4-fluoropiperidin-3-ylcarbamate

Method 2 was followed using (3R,4R)-benzyl3-(tert-butoxycarbonylamino)-4-fluoropiperidine-1-carboxylate (1.0equiv.) yielding crude tert-butyl(3R,4R)-4-fluoropiperidin-3-ylcarbamate, (93%). LCMS (m/z): 219.2 (MH⁺),LC R_(t)=0.45 min.

Synthesis of tert-butyl(3S,4S)-4-fluoropiperidin-3-ylcarbamate

Method 2 was followed using (3S,4S)-benzyl3-(tert-butoxycarbonylamino)-4-fluoropiperidine-1-carboxylate (1.0equiv.) yielding crude (±)-tert-butyl 4-fluoropiperidin-3-ylcarbamate,(93%). LCMS (m/z): 219.2 (MH⁺), LC R_(t)=0.45 min.

Synthesis oftert-butyl(3R,4R)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate

Method 2 was followed using (3R,4R)-benzyl3-(tert-butoxycarbonylamino)-4-(tert-butyldimethylsilyloxy)piperidine-1-carboxylate(1.0 equiv.) yielding crudetert-butyl(3R,4R)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate,(>99%). LCMS (m/z): 331.3 (MH⁺).

Synthesis oftert-butyl(3R,4R)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate

Method 2 was followed using (3S,4S)-benzyl3-(tert-butoxycarbonylamino)-4-(tert-butyldimethylsilyloxy)piperidine-1-carboxylate(1.0 equiv.) yielding crudetert-butyl(3S,4S)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate,(>99%). LCMS (m/z): 331.3 (MH⁺).

Synthesis oftert-butyl(3R,4R)-4-(tert-butyldimethylsilyloxy)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

Method 1 was followed using 1 eq each of 4-chloro-3-nitropyridine,tert-butyl (3R,4R)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamateand triethylamine in DMF yieldingtert-butyl(3R,4R)-4-(tert-butyldimethylsilyloxy)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate,(98%). LCMS (m/z): 453.3 (MH⁺); LC R_(t)=4.01 min.

Synthesis oftert-butyl(3S,4S)-4-(tert-butyldimethylsilyloxy)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

Method 1 was followed using 1 eq each of 4-chloro-3-nitropyridine,tert-butyl (3S,4S)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamateand triethylamine in DMF yieldingtert-butyl(3S,4S)-4-(tert-butyldimethylsilyloxy)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate,(98%). LCMS (m/z): 453.3 (MH⁺); LC R_(t)=4.01 min.

Synthesis oftert-butyl(3R,4R)-4-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

Method 1 of Example 1 was followed using 1 eq each of4-chloro-3-nitropyridine,tert-butyl(3R,4R)-4-fluoropiperidin-3-ylcarbamate and triethylamine inethanol yieldingtert-butyl(3R,4R)-4-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate,(91%). LCMS (m/z): 341.0 (MH⁺); LC R_(t)=2.37 min.

Synthesis oftert-butyl(3S,4S)-4-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

Method 1 of Example 1 was followed using 1 eq each of4-chloro-3-nitropyridine,tert-butyl(3S,4S)-4-fluoropiperidin-3-ylcarbamate and triethylamine inethanol yieldingtert-butyl(3S,4S)-4-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate,(91%). LCMS (m/z): 341.0 (MH⁺); LC R_(t)=2.37 min.

Synthesis oftert-butyl(3R,4R)-1-(3-aminopyridin-4-yl)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate

Following method 2,tert-butyl(3R,4R)-4-(tert-butyldimethylsilyloxy)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamatein ethanol and ethyl acetate (1:1, 0.1 M solution) was reduced yieldingtert-butyl(3R,4R)-1-(3-aminopyridin-4-yl)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate,(>99%). LCMS (m/z): 423.2 (MH⁺); LC R_(t)=3.78 min.

Synthesis oftert-butyl(3S,4S)-1-(3-aminopyridin-4-yl)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate

Following method 2,tert-butyl(3R,4R)-4-(tert-butyldimethylsilyloxy)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamatein ethanol and ethyl acetate (1:1, 0.1 M solution) was reduced yieldingtert-butyl(3R,4R)-1-(3-aminopyridin-4-yl)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate,(>99%). LCMS (m/z): 423.2 (MH⁺); LC R_(t)=3.78 min.

Synthesis oftert-butyl(3R,4R)-1-(3-aminopyridin-4-yl)-4-fluoropiperidin-3-ylcarbamate

Following method 2,tert-butyl(3R,4R)-4-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamatein ethanol and ethyl acetate (1:1, 0.1 M solution) was reduced yieldingtert-butyl(3R,4R)-1-(3-aminopyridin-4-yl)-4-fluoropiperidin-3-ylcarbamate,(>99%). LCMS (m/z): 311.2 (MH⁺); LC R_(t)=2.14 min.

Synthesis oftert-butyl(3S,4S)-1-(3-aminopyridin-4-yl)-4-fluoropiperidin-3-ylcarbamate

Following method 2,tert-butyl(3S,4S)-4-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamatein ethanol and ethyl acetate (1:1, 0.1 M solution) was reduced yieldingtert-butyl(3R,4R)-1-(3-aminopyridin-4-yl)-4-fluoropiperidin-3-ylcarbamate,(>99%). LCMS (m/z): 311.2 (MH⁺); LC R_(t)=2.14 min.

Synthesis oftert-butyl(3S,5R)-5-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate

tert-Butyl(3S,5R)-5-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamatewas prepared according to the patent procedure as described by Y, Zhou;WO2005028467.

Synthesis oftert-butyl(3S,5R)-5-(tert-butyldimethylsilyloxy)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

Method 1 was followed was followed usingtert-Butyl(3S,5R)-5-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate,yieldingtert-butyl(3S,5R)-5-(tert-butyldimethylsilyloxy)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate.LC/MS (m/z): 453.2 (MH⁺).

Synthesis oftert-butyl(3S,5R)-1-(3-aminopyridin-4-yl)-5-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate

Method 2 was followed usingtert-butyl(3S,5R)-5-(tert-butyldimethylsilyloxy)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate,yielding tert-butyl(3S,5R)-1-(3-aminopyridin-4-yl)-5-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate.LC/MS (m/z): 423.2 (MH⁺).

Synthesis of trans(±)-benzyl3-(bis(tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate

To a solution of trans(±)-benzyl3-(tert-butoxycarbonylamino)-4-hydroxypiperidine-1-carboxylate (1.0equiv.) in DCM and CH₃CN (1:1, 0.14 M) was added BOC₂O (1.0 equiv.),triethylamine (1.5 equiv.), and DMAP (catalytic amount). The reactionwas stirred at room temperature for 15 h, upon which time the solutionwas concentrated and purified via silica gel column chromatographyeluting with EtOAc and hexanes (1:6) to give the desired product as awhite foam. LCMS (m/z): 451.1 (MH⁺).

Synthesis of trans(±)-benzyl3-(bis(tert-butoxycarbonyl)amino)-4-methoxypiperidine-1-carboxylate

To a solution of NaH (1.3 equiv.) in THF (0.1M) was added benzyl3-(bis(tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate (1.0equiv.) and the reaction was heated to 50° C. for 10 min. Upon coolingto room temperature, MeI (1.5 equiv.) was added and the solution wasallowed to stir for 16 h. The reaction was quenched with water, thenextracted with EtOAc, the organic was dried with brine and Na₂SO₄, andconcentrated. The crude material was purified via silica gel columnchromatography eluting with EtOAc and hexanes (1:3) to give a clear oilin 71% yield. LCMS (m/z): 365.0 (MH⁺).

Synthesis oftrans(±)-3-(bis(tert-butoxycarbonyl)amino)-4-methoxypiperidine

Method 2 was followed using trans(±)-benzyl3-(bis(tert-butoxycarbonyl)amino)-4-methoxypiperidine-1-carboxylate (1.0equiv.) yielding crudetrans(±)-3-(bis(tert-butoxycarbonyl)amino)-4-methoxypiperidine that wasused for the next step without further purification. LCMS (m/z):331.2(MH⁺)

Synthesis oftrans(±)-N,N-di-BOC-4-methoxy-1-(3-nitropyridin-4-yl)piperidin-3-amine

Method 1 was followed usingtrans(±)-3-(bis(tert-butoxycarbonyl)amino)-4-methoxypiperidine (1.0equiv.), 4-chloro-3-nitropyridine (1.2 equiv.), and DIEA (4.0 equiv.) togivetrans(±)-N,N-di-BOC-4-methoxy-1-(3-nitropyridin-4-yl)-piperidin-3-amineafter column chromatography (EtOAc and hexanes, 50%) in 59% yield fortwo steps. LCMS (m/z): 453.2 (MH⁺), LC R_(t)=3.24 min.

Synthesis oftrans(±)-N,N-di-BOC-4-methoxy-1-(3-aminopyridin-4-yl)piperidin-3-amine

Method 2 was followed usingtrans(±)-N,N-di-BOC-4-methoxy-1-(3-nitropyridin-4-yl)-piperidin-3-amineto givetrans(±)-N,N-di-BOC-4-methoxy-1-(3-aminopyridin-4-yl)piperidin-3-aminein >95% yield as a clear oil. LCMS (m/z): 423.0 (MH⁺), LC R_(t)=3.10min.

Synthesis of of (3R,5R)-5-(tert-butyldimethylsilyloxy)piperidin-3-ol

(3R,5R)-5-(tert-butyldimethylsilyloxy)piperidin-3-ol was preparedaccording to the patent procedure as described by Zhou, Y. WO2005028467.

Synthesis of (3R,5R)-benzyl3-(tert-butyldimethylsilyloxy)-5-hydroxypiperidine-1-carboxylate

To a solution of (3R,5R)-5-(tert-butyldimethylsilyloxy)piperidin-3-ol (1eq) in 20 mL of 1,4-dioxane and 8 mL of water was added benzylchloroformate (1.5 eq). The mixture was stirred at room temperature for4 hours. The crude mixture was diluted with 100 mL of EtOAc, washed withbrine, then dried over anhydrous MgSO₄, filtered, and concentrated invacuo. The crude residue was purified by flash chromatography(EtOAc:hexanes=1:3) to yield (3R,5R)-benzyl3-(tert-butyldimethylsilyloxy)-5-hydroxypiperidine-1-carboxylate (74%).LC/MS (m/z): 366.2 (MH⁺).

Synthesis of (3S,5R)-benzyl3-(benzoyloxy)-5-(tert-butyl-dimethylsilyloxy)piperidine-1-carboxylate

To a stirring 0° C. solution of triphenylphosphine (1.2 equiv) in 23 mLof THF was added di-tert-butyl azodicarboxylate (1.2 equiv). The mixturewas stirred at 0° C. for 10 minutes. Then a solution of (3R,5R)-benzyl3-(tert-butyldimethylsilyloxy)-5-hydroxypiperidien-1-carboxylate (1.0equiv) in 11 mL of THF was added and stirred for 20 minutes at 0° C.Benzoic acid (1.2 equiv) was then added and the reaction mixture wasallowed to slowly warm to rt. After 16 hours the reaction mixture wasconcentrated in vacuo then diluted with EtOAc and washed with water thenbrine. The organic layer was dried with Na₂SO₄, filtered, andconcentrated in vacuo. The crude residue was purified by flashchromatography (EtOAc:hexanes=1:8) to yield (3S,5R)-benzyl3-(benzoyloxy)-5-(tert-butyl-dimethylsilyloxy)piperidine-1-carboxylate(77%). LC/MS (m/z): 470.2 (MH⁺), HPLC R_(t)=6.05 min.

Synthesis of (3S,5R)-benzyl3-(benzoyloxy)-5-hydroxypiperidine-1-carboxylate

To a solution of (3S,5R)-benzyl3-(benzoyloxy)-5-(tert-butyl-dimethylsilyloxy)-piperidine-1-carboxylate(1 eq) in 30 mL of methanol was added 3.8M HCl in isopropanol (4 eq).The reaction mixture was allowed to stand at room temperature for 3hours at which point it was concentrated under reduced pressure. Theresulting residue was diluted with 120 mL of EtOAc, washed with sat. aq.sodium bicarbonate, brine, then dried over anhydrous MgSO₄, filtered,and concentrated in vacuo. The crude residue was purified by flashchromatography (EtOAc:hexanes=1:1) to yield (3S,5R)-benzyl3-(benzoyloxy)-5-hydroxypiperidine-1-carboxylate (95%). LC/MS (m/z):355.9 (MH⁺). HPLC: R_(t): 3.62 min.

Synthesis of (3S,5S)-benzyl3-azido-5-(benzoyloxy)piperidine-1-carboxylate

To a solution of (3S,5R)-benzyl3-(benzoyloxy)-5-hydroxypiperidine-1-carboxylate (1 eq) in 20 mL ofdichloromethane was added triethyl amine (3 eq) and methanesulfonylchloride (1.5 eq) at 0° C. The reaction mixture was allowed to stir atroom temperature for 2 hours. The crude mixture was diluted with 120 mLof EtOAc, washed with sat. aq. sodium bicarbonate, brine, then driedover anhydrous MgSO₄, filtered, and concentrated in vacuo. The cruderesidue was dissolved in 25 mL of NMP. Sodium azide (2.2 eq) was addedand the resulting suspension was stirred at 80° C. overnight. Thereaction mixture was diluted with 200 mL of EtOAc and 100 mL of hexanes,washed with water, brine, then dried over anhydrous MgSO₄, filtered, andconcentrated in vacuo. The crude residue was purified by flashchromatography (EtOAc:hexanes=1:2) to yield (3S,5S)-benzyl3-azido-5-(benzoyloxy)piperidine-1-carboxylate (88%). LC/MS (m/z): 381.0(MH⁺). HPLC: R_(t): 4.41 min.

Synthesis of (3S,5S)-benzyl3-(benzoyloxy)-5-(tert-butoxycarbonylamino)-piperidine-1-carboxylate

To a solution of (3S,5S)-benzyl3-azido-5-(benzoyloxy)piperidine-1-carboxylate (1 eq) in a mixture of 14mL of pyridine and 2 mL of ammonium hydroxide was added 1Mtrimethylphosphine (3 eq) at room temperature. The reaction mixture wasstirred at room temperature for 3 hours at which point the solvents wereremoved under reduced pressure to give a yellow oil. The oil was againdissolved in 100 mL of ethanol and concentrated to remove ammoniumhydroxide completely. The residue was dissolved in 24 ml of 1,4-dioxaneand 24 mL of sat. aq. NaHCO₃ was added. Di-tert-butyl dicarbonate (4 eq)in 12 mL of THF was added dropwise at 0° C. The mixture was allowed tostir at room temperature for 2 hours. The crude mixture was diluted with200 mL of EtOAc, washed with brine, then dried over anhydrous MgSO₄;filtered, and concentrated in vacuo. The crude residue was purified byflash chromatography (EtOAc:hexanes=1:2) to yield (3S,5S)-benzyl3-(benzoyloxy)-5-(tert-butoxycarbonylamino)-piperidine-1-carboxylate(92%). LC/MS (m/z): 455.1 (MH⁺). HPLC: R_(t): 4.38 min.

Synthesis of(3S,5S)-5-(tert-butoxycarbonylamino)-1-(3-nitropyridin-4-yl)-piperidin-3-ylbenzoate

To a solution of (3S,5S)-benzyl3-(benzoyloxy)-5-(tert-butoxycarbonylamino)-piperidine-1-carboxylate (1eq) in 15 methanol and 15 mL of EtOAc was added 10% Pd/C (0.1 eq). Theresulting suspension was stirred at H₂ atmosphere for 4 hours. The crudesolids were filtered through a pad of Celite on a paper lined Buchnerfunnel, washed with MeOH, then concentrated in vacuo. The residue wasdissolved in 20 mL of isopropanol and DIPEA (1.8 eq) and4-chloro-3-nitropyridine (1.2 eq) were added. The reaction mixture wasstirred at 75° C. for 2 hours, at which point the reaction mixture wasallowed to cool to room temperature and concentrated under reducedpressure. The residue was diluted with 150 mL of EtOAc, washed withbrine, then dried over anhydrous MgSO₄, filtered, and concentrated invacuo. The crude residue was purified by flash chromatography(EtOAc:hexanes=1:1) to yield(3S,5S)-5-(tert-butoxycarbonylamino)-1-(3-nitropyridin-4-yl)-piperidin-3-ylbenzoate (90%). LC/MS (m/z): 443.2 (MH⁺). HPLC: R_(t): 2.89 min.

Synthesis of(3S,5S)-1-(3-aminopyridin-4-yl)-5-(tert-butoxycarbonylamino)piperidin-3-ylbenzoate

Following Method 2,(3S,5S)-5-(tert-butoxycarbonylamino)-1-(3-nitropyridin-4-yl)-piperidin-3-ylbenzoate was reduced to yield(3S,5S)-1-(3-aminopyridin-4-yl)-5-(tert-butoxycarbonylamino)piperidin-3-ylbenzoate. LC/MS (m/z): 413.1 (MH⁺). HPLC: R_(t): 2.75 min.

Synthesis of (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-(tert-butyldimethylsilyloxy)piperidine-1-carboxylate

Followed method to synthesis of (3S,5S)-benzyl3-(benzoyloxy)-5-(tert-butoxycarbonylamino)-piperidine-1-carboxylatestarting from (3R,5R)-benzyl3-(tert-butyldimethylsilyloxy)-5-hydroxypiperidine-1-carboxylate. LC/MS(m/z): 365.2 (MH⁺-Boc), Rt: 1.37.

Synthesis of (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-hydroxypiperidine-1-carboxylate

To a solution of (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-(tert-butyldimethylsilyloxy)piperidine-1-carboxylate(1 eq) in 30 mL of THF was added 5.2 mL of TBAF (1.2 eq). The reactionmixture was stirred at room temperature for 2 hours. The mixture wasdiluted with ethyl acetate, washed with brine, then dried over anhydrousMgSO₄, filtered, and concentrated in vacuo. The crude residue waspurified by flash chromatography (5% methanol in EtOAc:hexanes=1:1) toyield (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-hydroxypiperidine-1-carboxylate (100%).LC/MS (m/z): 251.2 (MH⁺), Rt: 0.89. HPLC: Rt: 3.26 min.

Synthesis of (3S 5S)-benzyl3-(tert-butoxycarbonylamino)-5-fluoropiperidine-1-carboxylate

To a solution of the (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-hydroxypiperidine-1-carboxylate (1 eq) in5 mL of dichloromethane was added DAST (1.35 eq). The reaction mixturewas stirred at room temperature for 2 hours. The mixture was dilutedwith 120 mL of ethyl acetate, washed with brine, then dried overanhydrous MgSO₄, filtered, and concentrated in vacuo. The crude residuewas purified by flash chromatography (EtOAc:hexanes=1:3) to yieldtert-butyl(3S,5S)-1-(3-aminopyridin-4-yl)-5-fluoropiperidin-3-ylcarbamate(30%). LC/MS (m/z): 253.1 (MH⁺-100), R_(t)=0.96 min. HPLC: R_(t): 3.79min.

Synthesis oftert-butyl(3S,5S)-5-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

To a solution of (3S,5S)-benzyl3-(tert-butoxycarbonylamino)-5-fluoropiperidine-1-carboxylate (1 eq) in5 methanol and 5 mL of EtOAc was added 10% Pd/C (0.1 eq). The resultingsuspension was stirred at H₂ atmosphere for 4 hours. The crude solidswere filtered through a pad of Celite on a paper lined Buchner funnel,washed with MeOH, then concentrated in vacuo. The residue was dissolvedin 5 mL of isopropanol and DIPEA (1.8 eq) and 4-chloro-3-nitropyridine(1.5 eq) were added. The reaction mixture was stirred at 65° C. for 3hours, at which point the reaction mixture was allowed to cool to roomtemperature and concentrated under reduced pressure. The residue wasdiluted with 120 mL of EtOAc, washed with brine, then dried overanhydrous MgSO₄, filtered, and concentrated in vacuo. The crude residuewas purified by flash chromatography (EtOAc:hexanes=1:1) to givetert-butyl(3S,5S)-5-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate(78%). LC/MS (m/z): 341.1 (MH⁺), R_(t)=0.57 min. HPLC: R_(t): 2.01 min.

Synthesis oftert-butyl(3S,5S)-1-(3-aminopyridin-4-yl)-5-fluoropiperidin-3-ylcarbamate

Following Method 2,tert-butyl(3S,5S)-5-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamatewas reduced to yieldtert-butyl(3S,5S)-1-(3-aminopyridin-4-yl)-5-fluoropiperidin-3-ylcarbamate.LC/MS (m/z): 311.1 (MH⁺), R_(t)=0.54 min. HPLC: R_(t): 1.76 min.

Synthesis of (S)-benzyl3-(tert-butoxycarbonylamino)-5-oxopiperidine-1-carboxylate

To a solution of (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-hydroxypiperidine-1-carboxylate (1 eq) in10 mL dichloromethane was added Dess-Martin periodinane (1.2 eq) at roomtemperature. The reaction mixture was stirred at that temperatureovernight. The mixture was diluted with ethyl acetate, washed withbrine, then dried over anhydrous MgSO₄, filtered, and concentrated invacuo. The crude residue was purified by flash chromatography(EtOAc:hexanes=1:2) to yield (S)-benzyl3-(tert-butoxycarbonylamino)-5-oxopiperidine-1-carboxylate (81%). LC/MS(m/z): 249.1 (MH⁺-100), R_(t): 0.83 min. HPLC: R_(t): 3.26 min.

Synthesis of5-(tert-butoxycarbonylamino)-3,3-difluoropiperidine-1-carboxylate

To a solution of the (S)-benzyl3-(tert-butoxycarbonylamino)-5-oxopiperidine-1-carboxylate (1 eq) in 25mL of dichloromethane was added DAST (20 eq). The reaction mixture wasstirred at room temperature 3 hours. The reaction was quenched by aq.sodium bicarbonate. The resulting mixture was extracted with ethylacetate. The organic layer was separated and washed with brine, thendried over anhydrous MgSO₄, filtered, and concentrated in vacuo. Thecrude residue was purified by flash chromatography (EtOAc:hexanes=1:2)to yield5-(tert-butoxycarbonylamino)-3,3-difluoropiperidine-1-carboxylate (52%).LC/MS (m/z): 271.1 (-Boc), R_(t): 0.99 min.

Synthesis of (S)-tert-butyl5,5-difluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

To a solution of5-(tert-butoxycarbonylamino)-3,3-difluoropiperidine-1-carboxylate (1 eq)in 5 mL of methanol and 5 mL of EtOAc was added 10% Pd/C (0.1 eq). Theresulting suspension was stirred at H2 atmosphere overnight. The crudesolids were filtered through a pad of Celite on a paper lined Buchnerfunnel, washed with MeOH, then concentrated in vacuo. The residue wasdissolved in 5 mL of isopropanol and DIPEA (2.0 eq) and4-chloro-3-nitropyridine (1.5 eq) were added. The reaction mixture wasstirred at 70° C. for 3 hours, at which point the reaction mixture wasallowed to cool to room temperature and concentrated under reducedpressure. The residue was diluted with 120 mL of EtOAc, washed withbrine, then dried over anhydrous MgSO₄, filtered, and concentrated invacuo. The crude residue was purified by flash chromatography (5%methanol in EtOAc:hexanes=1:1) to yield (S)-tert-butyl5,5-difluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate (19%). LC/MS(m/z): 359.0 (MH⁺), R_(t): 0.65 min.

Synthesis of (S)-tert-butyl1-(3-aminopyridin-4-yl)-5,5-difluoropiperidin-3-ylcarbamate

Following Method 2, (S)-tert-butyl5,5-difluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate was reducedto yield (S)-tert-butyl1-(3-aminopyridin-4-yl)-5,5-difluoropiperidin-3-ylcarbamate. LC/MS(m/z): 329.0 (MH⁺), R_(t): 0.62 min.

Synthesis of (S)-benzyl3-(tert-butoxycarbonylamino)-5-ethylidenepiperidine-1-carboxylate

To a suspension of ethyltriphenylphosphonium bromide (11 eq) in 14 mL ofTHF was added potassium tert-butoxide (10 eq) at room temperature. Thereaction mixture was stirred at that temperature for 20 minutes. Thenthe reaction was cooled to 0° C., and (S)-benzyl3-(tert-butoxycarbonylamino)-5-oxopiperidine-1-carboxylate (1 eq) in 7mL of THF was added to the reaction mixture. The reaction was allowed towarm up room temperature. After being stirred for 40 minutes, thereaction mixture was poured into aq. sodium bicarbonate and extractedwith ethyl acetate. The organic layer was separated and washed withbrine, then dried over anhydrous MgSO₄, filtered, and concentrated invacuo. The crude residue was purified by flash chromatography(EtOAc:hexanes=2:1) to yield (S)-benzyl3-(tert-butoxycarbonylamino)-5-ethylidenepiperidine-1-carboxylate. LC/MS(m/z): 261.2 (MH⁺-100), R_(t): 1.12 min. HPLC: R_(t): 4.31 min.

Synthesis oftert-butyl(3S,5R)-5-ethyl-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

To a solution of (S)-benzyl3-(tert-butoxycarbonylamino)-5-ethylidenepiperidine-1-carboxylate (1 eq)in 5.5 mL of ethanol and 5.5 mL of EtOAc was added 10% Pd/C (0.1 eq).The resulting suspension was stirred at H₂ atmosphere for 45 minutes.The crude solids were filtered through a pad of Celite on a paper linedBuchner funnel, washed with MeOH, then concentrated in vacuo. Theresidue was dissolved in 1.4 mL of isopropanol and DIPEA (2.5 eq) and4-chloro-3-nitropyridine (1.5 eq) were added. The reaction mixture wasstirred at 80° C. overnight, at which point the reaction mixture wasallowed to cool to room temperature and concentrated under reducedpressure. The residue was diluted with 120 mL of EtOAc, washed withbrine, then dried over anhydrous MgSO₄, filtered, and concentrated invacuo. The crude residue was purified by flash chromatography(EtOAc:hexanes=1:1) to yieldtert-butyl(3S,5R)-5-ethyl-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate(91%). LC/MS (m/z): 351.2 (MH⁺), R_(t): 0.75 min.

Synthesis oftert-butyl(3S,5R)-1-(3-aminopyridin-4-yl)-5-ethylpiperidin-3-ylcarbamate

Following Method 2,tert-butyl(3S,5R)-5-ethyl-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamatewas reduced to yieldtert-butyl(3S,5R)-1-(3-aminopyridin-4-yl)-5-ethylpiperidin-3-ylcarbamate.LC/MS (m/z): 321.2 (MH⁺), R_(t): 0.73 min. HPLC: R_(t): 2.65 min.

Synthesis of (3R,5R)-benzyl3-(tert-butyldimethylsilyloxy)-5-methoxypiperidine-1-carboxylate

To a solution of (3R,5R)-benzyl3-(tert-butyldimethylsilyloxy)-5-hydroxypiperidine-1-carboxylate (1 eq)in 30 mL of THF was added sodium hydride (1.5 eq) and followed by methyliodide (5 eq) at 0° C. The reaction mixture was allowed to stir at roomtemperature for 3 hours. The crude mixture was diluted with 120 mL ofEtOAc, washed with brine, then dried over anhydrous MgSO₄, filtered, andconcentrated in vacuo. The crude residue was purified by flashchromatography (EtOAc:hexanes=1:5) to yield (3R,5R)-benzyl3-(tert-butyldimethylsilyloxy)-5-methoxypiperidine-1-carboxylate (93%).LC/MS (m/z): 380.2 (MH⁺).

Synthesis of (3R,5R)-benzyl 3-hydroxy-5-methoxypiperidine-1-carboxylate

To a solution of (3R,5R)-benzyl3-(tert-butyldimethylsilyloxy)-5-methoxypiperidine-1-carboxylate (1 eq)in 30 mL of methanol was added 3.8M HCl in isopropanol (4 eq). Thereaction mixture was allowed to stand at room temperature for 3 hours atwhich point it was concentrated under reduced pressure. The resultingresidue was diluted with 100 mL of EtOAc, washed with sat. aq. sodiumbicarbonate, brine, then dried over anhydrous MgSO₄, filtered, andconcentrated in vacuo. The crude residue was purified by flashchromatography (EtOAc:hexanes=2:1) to yield (3R,5R)-benzyl3-hydroxy-5-methoxypiperidine-1-carboxylate (92%). LC/MS (m/z): 266.2(MH⁺).

Synthesis of (3S,5R)-benzyl 3-azido-5-methoxypiperidine-1-carboxylate

To a solution of (3R,5R)-benzyl3-hydroxy-5-methoxypiperidine-1-carboxylate (1 eq) in 40 mL ofdichloromethane was added triethyl amine (3 eq) and methanesulfonylchloride (1.5 eq) at 0° C. The reaction mixture was allowed to stir atroom temperature for 2 hours. The crude mixture was diluted with 150 mLof EtOAc, washed with sat. aq. sodium bicarbonate, brine, then driedover anhydrous MgSO₄, filtered, and concentrated in vacuo. The cruderesidue was purified by flash chromatography (EtOAc:hexanes=1:1) to givethe intermediate, which was dissolved in 15 mL of DMF. Sodium azide (3.3eq) was added and the resulting suspension was stirred at 80° C.overnight. The reaction mixture was diluted with 150 mL of EtOAc, washedwith water, brine, then dried over anhydrous MgSO₄, filtered, andconcentrated in vacuo. The crude residue was purified by flashchromatography (EtOAc:hexanes=1:2) to yield (3S,5R)-benzyl3-azido-5-methoxypiperidine-1-carboxylate (95%). LC/MS (m/z): 263.2(MH⁺-28).

Synthesis of (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-methoxypiperidine-1-carboxylate

To a solution of (3S,5R)-benzyl3-azido-5-methoxypiperidine-1-carboxylate (1 eq) in a mixture of 14 mLof pyridine and 2 mL of ammonium hydroxide was added 1Mtrimethylphosphine (3 eq) at room temperature. The reaction mixture wasstirred at room temperature for 4 hours at which point the solvents wereremoved under reduced pressure to give a yellow oil. The oil was againdissolved in 100 mL of ethanol and concentrated to remove ammoniumhydroxide completely. The residue was dissolved in 16 ml of 1,4-dioxaneand 16 mL of sat. aq. NaHCO₃ was added. Di-tert-butyl dicarbonate (4 eq)in 8 mL of THF was added dropwise at 0° C. The mixture was allowed tostir at room temperature for 2 hours. The crude mixture was diluted with300 mL of EtOAc, washed with brine, then dried over anhydrous MgSO₄,filtered, and concentrated in vacuo. The crude residue was purified byflash chromatography (EtOAc:hexanes=1:1) to yield (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-methoxypiperidine-1-carboxylate (86%).LC/MS (m/z): 365.0 (MH⁺).

Synthesis oftert-butyl(3S,5R)-5-methoxy-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

To a solution of (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-methoxypiperidine-1-carboxylate (1 eq) in25 methanol was added 10% Pd/C (0.1 eq). The resulting suspension wasstirred at H₂ atmosphere for 2 hours. The crude solids were filteredthrough a pad of Celite on a paper lined Buchner funnel, washed withMeOH, then concentrated in vacuo. The residue was dissolved in 25 mL ofisopropanol and DIEA (1.8 eq) and 4-chloro-3-nitropyridine (1.2 eq) wereadded. The reaction mixture was stirred at 80° C. for 4 hours, at whichpoint the reaction mixture was allowed to cool to room temperature andconcentrated under reduced pressure. The residue was diluted with 150 mLof EtOAc, washed with brine, then dried over anhydrous MgSO₄, filtered,and concentrated in vacuo. The crude residue was purified by flashchromatography (5% methanol in EtOAc:hexanes=1:1) to yield(3S,5R)-5-methoxy-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate (88%).LC/MS (m/z): 353.0 (MH⁺). HPLC: R_(t): 2.15 min.

Synthesis oftert-Butyl(3S,5R)-1-(3-aminopyridin-4-yl)-5-methoxypiperidin-3-ylcarbamate

Following Method 2,tert-butyl(3S,5R)-5-methoxy-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamatewas reduced yieldingtert-Butyl(3S,5R)-1-(3-aminopyridin-4-yl)-5-methoxypiperidin-3-ylcarbamate.LC/MS (m/z): 323.1 (MH⁺).

Synthesis oftert-butyl(3S,5R)-1-(3-aminopyridin-4-yl)-5-ethoxypiperidin-3-ylcarbamate

The procedure to prepare this compound is as same as the methoxycompound. LC/MS (m/z): 337.1 (MH⁺), R_(t): 0.63 min. HPLC: R_(t): 2.47min.

Synthesis of(3R,5R)-3-(tert-butyldimethylsilyloxy)-5-fluoro-1-(4-methoxybenzyl)piperidine

(3R,5R)-3-(tert-butyldimethylsilyloxy)-5-fluoro-1-(4-methoxybenzyl)-piperidinewas prepared according to the literature procedure as described byCossy, J. Synlett, 2007, 263.

Synthesis of (3R,5R)-3-(tert-butyldimethylsilyloxy)-5-fluoropiperidine

To a solution of(3R,5R)-3-(tert-butyldimethylsilyloxy)-5-fluoro-1-(4-methoxybenzyl)piperidine(1 eq) in 5 mL of methanol was added 10% Pd/C (0.2 eq). The resultingsuspension was stirred at H₂ atmosphere overnight. The crude solids werefiltered through a pad of Celite on a paper lined Buchner funnel, washedwith MeOH, then concentrated in vacuo to yield(3R,5R)-3-(tert-butyldimethylsilyloxy)-5-fluoropiperidine, which wasused to next step without further purification. LC/MS (m/z): 234.1(MH⁺).

Synthesis of (3R,5R)-benzyl 3-fluoro-5-hydroxypiperidine-1-carboxylate

To a solution of(3R,5R)-3-(tert-butyldimethylsilyloxy)-5-fluoropiperidine (1 eq) in 30mL of methanol was added 3.8M HCl in isopropanol (4 eq). The reactionmixture was allowed to stand at room temperature for 3 hours at whichpoint it was concentrated under reduced pressure. The resulting residuewas diluted with 120 mL of EtOAc, washed with sat. aq. sodiumbicarbonate, brine, then dried over anhydrous MgSO₄, filtered, andconcentrated in vacuo. The crude residue was purified by flashchromatography (EtOAc:hexanes=2:1) to give (3R,5R)-benzyl3-fluoro-5-hydroxypiperidine-1-carboxylate, (94%). LC/MS (m/z): 254.2(MH⁺).

Synthesis of (3S,5R)-benzyl 3-azido-5-fluoropiperidine-1-carboxylate

To a solution of (3R,5R)-benzyl3-fluoro-5-hydroxypiperidine-1-carboxylate (1 eq) in 14 mL ofdichloromethane was added triethyl amine (3 eq) and methanesulfonylchloride (1.5 eq) at 0° C. The reaction mixture was allowed to stir atroom temperature for 1.5 hours. The crude mixture was diluted with 120mL of diethyl ether, washed with sat. aq. sodium bicarbonate, brine,then dried over anhydrous MgSO₄, filtered, and concentrated in vacuo.The crude residue was dissolved in 16 mL of NMP. Sodium azide (3.0 eq)was added and the resulting suspension was stirred at 80° C. overnight.The reaction mixture was diluted with 200 mL of EtOAc and 100 mL ofhexanes, washed with water, brine, then dried over anhydrous MgSO₄,filtered, and concentrated in vacuo. The crude residue was purified byflash chromatography (EtOAc:hexanes=1:3) to yield (3S,5R)-benzyl3-azido-5-fluoropiperidine-1-carboxylate (90%). LC/MS (m/z): 251.1(MH⁺-28).

Synthesis of (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-fluoropiperidine-1-carboxylate

To a solution of (3S,5R)-benzyl 3-azido-5-fluoropiperidine-1-carboxylate(1 eq) in a mixture of 11 mL of pyridine and 1.5 mL of ammoniumhydroxide was added 1M trimethylphosphine (3 eq) at room temperature.The reaction mixture was stirred at room temperature for 3 hours atwhich point the solvents were removed under reduced pressure to give ayellow oil. The oil was again dissolved in 100 mL of ethanol andconcentrated to remove ammonium hydroxide completely. The residue wasdissolved in 12 ml of 1,4-dioxane and 12 mL of sat. aq. NaHCO₃ wasadded. Di-tert-butyl dicarbonate (4 eq) in 6 mL of THF was addeddropwise at 0° C. The mixture was allowed to stir at room temperaturefor 1 hour. The crude mixture was diluted with 150 mL of EtOAc, washedwith brine, then dried over anhydrous MgSO₄, filtered, and concentratedin vacuo. The crude residue was purified by flash chromatography(EtOAc:hexanes=1:1) to yield (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-fluoropiperidine-1-carboxylate (95%).LC/MS (m/z): 253.1 (MH⁺-100).

Synthesis oftert-butyl(3S,5R)-5-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

To a solution of (3S,5R)-benzyl3-(tert-butoxycarbonylamino)-5-fluoropiperidine-1-carboxylate (1 eq) in28 methanol was added 10% Pd/C (0.1 eq). The resulting suspension wasstirred at H₂ atmosphere for 1 hours. The crude solids were filteredthrough a pad of Celite on a paper lined Buchner funnel, washed withMeOH, then concentrated in vacuo. The residue was dissolved in 33 mL ofisopropanol and DIPEA (2.5 eq) and 4-chloro-3-nitropyridine (1.5 eq)were added. The reaction mixture was stirred at 80° C. for 2 hours, atwhich point the reaction mixture was allowed to cool to room temperatureand concentrated under reduced pressure. The residue was diluted with150 mL of EtOAc, washed with brine, then dried over anhydrous MgSO₄,filtered, and concentrated in vacuo. The crude residue was purified byflash chromatography (5% methanol in EtOAc:hexanes=1:1) to yieldtert-butyl(3S,5R)-5-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate(90%). LC/MS (m/z): 341.1 (MH⁺). HPLC: R_(t): 2.12 min.

Synthesis oftert-Butyl(3S,5R)-1-(3-aminopyridin-4-yl)-5-fluoropiperidin-3-ylcarbamate

Following Method 2,tert-butyl(3S,5R)-5-fluoro-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamatewas reduced yieldingtert-Butyl(3S,5R)-1-(3-aminopyridin-4-yl)-5-fluoropiperidin-3-ylcarbamate.LC/MS (m/z): 311.1 (MH⁺).

Synthesis of tert-butyl 5-methylpyridin-3-ylcarbamate

To a solution of 5-methylpyridin-3-amine (5 g, 46 mmol) in THF (80 mL)at r.t. was added 1M sodium bis(trimethylsilylamide) in THF (101 mL, 101mmol), stirred for 15 min, followed by di-tert-butyldicarbonate (11 g,49 mmol) in THF (20 mL). The reaction was stirred at r.t overnight andconcentrated. The concentrate was treated with 0.2M HCl (60 mL) andEtOAc, and the organic layer was extracted, washed with NaHCO_(3(sat.))and brine, dried over Na₂SO₄, filtered and concentrated. The concentratewas purified using flash chromatography on silica gel (40% EtOAc:Hexane)to give a yellow solid as product tert-butyl5-methylpyridin-3-ylcarbamate (8.5 g, 88% yield). LCMS (m/z): 209.1(MH⁺); LC R_(t)=1.94 min. ¹H NMR(CDCl₃) δ 8.20(d, 1H), 8.12(s, 1H),7.86(s, 1H), 6.53(s, 1H), 2.33(s, 3H), 1.53(s, 9H).

Synthesis of cis-(±)-tert-butyl 5-methylpiperidin-3-ylcarbamate

To a solution of 5-methylpyridin-3-ylcarbamate (3 g, 14 mmol) in glacialacetic Acid (50 mL) was added 5% Rhodium on active carbon (0.5 g) andPlatinum(IV) oxide (0.5 g) in the hydrogenation steel bomb. The mixturewas sealed and hydrogenated at 200 psi and 70° C. for 48 hours. Themixture was filtered through Celite and concentrated to givecis-(±)-tert-butyl 5-methylpiperidin-3-ylcarbamate. LCMS (m/z): 215.1(MH⁺).

Synthesis of cis-(±)-tert-butyl5-methyl-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

Method 1 was followed using crude cis-(±)-tert-butyl5-methylpiperidin-3-ylcarbamate yielding cis-(±)-tert-butyl5-methyl-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate (66% yield).LCMS (m/z): 337.1 (MH⁺); LC R_(t)=2.50 min. ¹H NMR(CDCl₃) δ 8.84(s, 1H),8.36(d, 1H), 7.04(m, 1H), 4.44(m, 1H), 3.90(m, 1H), 3.71(m, 1H), 3.09(d,1H), 2.66(q, 2H), 2.10(d, 1H), 1.84(m, 1H), 1.56(s, 9H), 0.93(d, 3H).

Synthesis of cis-(±)-tert-butyl1-(3-aminopyridin-4-yl)-5-methylpiperidin-3-ylcarbamate

Method 2 was followed using cis-(±)-tert-butyl5-methyl-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate yieldingcis-(±)-tert-butyl5-methyl-1-(3-aminopyridin-4-yl)piperidin-3-ylcarbamate (98% yield).LCMS (m/z): 307.1 (MH⁺); LC R_(t)=2.44 min. ¹H NMR(CDCl₃) δ 8.01(s, 1H),7.95(d, 1H), 6.76(d, 1H), 4.40(m, 1H), 3.70(m, 3H), 3.58(dq, 1H),3.21(dq, 1H), 2.15(m, 3H), 1.90(m, 1H), 1.58(s, 9H), 0.97(d, 3H).

Synthesis of tert-butyl 5-(trifluoromethyl)pyridin-3-ylcarbamate

To a solution of 5-trifluoromethylpyridin-3-amine (1 eq.) in THF (80 mL)at r.t. was added 1M sodium bis(trimethylsilylamide) in THF (2 eq.),stirred for 15 min, followed by di-tert-butyldicarbonate (1 eq.) in THF.The reaction was stirred at r.t overnight and concentrated. Theconcentrate was treated with 0.2M HCl aq. and EtOAc, and the organiclayer was extracted, washed with NaHCO_(3(sat.)) and brine, dried overNa₂SO₄, filtered and concentrated. The concentrate was purified usingflash chromatography on silica gel (40% EtOAc:Hexane) to give a yellowsolid as product tert-butyl 5-(trifluoromethyl)pyridin-3-ylcarbamate(31% yield). LCMS (m/z): 263.0 (MH⁺); LC R_(t)=3.84 min. ¹H NMR(CDCl₃) δ8.56(m, 2H), 8.34(s, 1H), 6.71 (s, 1H), 1.55 (s, 9H).

Synthesis of cis-(±)-tert-butyl5-(trifluoromethyl)piperidin-3-ylcarbamate

To a solution of tert-butyl 5-(trifluoromethyl)pyridin-3-ylcarbamate (3g, 14 mmol) in glacial acetic acid (50 mL) was added 5% Rhodium onactive carbon (0.5 g) and Platinum(IV) oxide (0.5 g) in thehydrogenation steel bomb. The mixture was sealed and hydrogenated at 200psi and 70° C. for 48 h. the mixture was filtered through Celite andconcentrated to give cis-(±)-tert-butyl5-(trifluoromethyl)piperidin-3-ylcarbamate. LCMS (m/z): 269.1 (MH⁺).

Synthesis of cis-(±)-tert-butyl1-(3-nitropyridin-4-yl)-5-(trifluoromethyl)piperidin-3-ylcarbamate

Method 1 was followed using crude cis-(±)-tert-butyl5-(trifluoromethyl)piperidin-3-ylcarbamate yielding cis-(±)-tert-butyl1-(3-nitropyridin-4-yl)-5-(trifluoromethyl) piperidin-3-ylcarbamate (42%yield over two steps). LCMS (m/z): 391.1 (MH⁺); LC R_(t)=2.92 min. ¹HNMR(CDCl₃) δ 8.93(s, 1H), 8.47(d, 1H), 7.01(d, 1H), 4.50(m, 1H), 3.80(m,2H), 3.45(m, 1H), 3.00(t, 1H), 2.66(m, 1H), 2.63(m, 1H), 2.38(d, 1H),1.56(s, 9H).

Synthesis cis-(±)-tert-butyl1-(3-aminopyridin-4-yl)-5-(trifluoromethyl)piperidin-3-ylcarbamate

Method 2 was followed using cis-(±)-tert-butyl1-(3-nitropyridin-4-yl)-5-(trifluoromethyl)piperidin-3-ylcarbamateyielding cis-(±)-tert-butyl1-(3-aminopyridin-4-yl)-5-(trifluoromethyl)piperidin-3-ylcarbamate. LCMS(m/z): 361.0 (MH⁺); LC R_(t)=2.72 min. ¹H NMR(CDCl₃) δ 8.05(s, 1H),7.98(d, 1H), 6.79 (d, 1H), 4.46(m, 1H), 3.83(m, 1H), 3.72(s, 2H),3.62(m, 1H), 3.49(m, 1H), 2.59(m, 2H), 2.36(m, 1H), 2.23(t, 1H), 1.58(s,9H).

Synthesis of cis(±)-1-benzyl 3-methyl5-(tert-butoxycarbonylamino)piperidine-1,3-dicarboxylate

To a solution ofcis(±)-1-(benzyloxycarbonyl)-5-(tert-butoxycarbonylamino)piperidine-3-carboxylicacid (1.0 eq), methanol (20 eq.) and EDC (1.3 eq) in dichloromethane ata concentration of 0.25 M at 0° C. was added dimethylaminopyridine (0.1eq). After stirring for 48 hours as the reaction was allowed to warm tort the volatiles were removed in vacuo. Upon addition of ethyl acetateand washing with H₂O (3×), 1N HCl, NaHCO_(3(sat.)) and brine, thesolution was dried over MgSO₄, filtered, concentrated and purified bycolumn chromatography (25% ethyl acetate/hexanes) to yieldcis(±)-1-benzyl 3-methyl5-(tert-butoxycarbonylamino)-piperidine-1,3-dicarboxylate. LCMS (m/z):293.1 (MH-Boc⁺); LC R_(t)=4.09 min

Synthesis of cis(±)-benzyl3-(tert-butoxycarbonylamino)-5-(hydroxymethyl)piperidine-1-carboxylate

A solution of cis(±)-1-benzyl 3-methyl5-(tert-butoxycarbonylamino)piperidine-1,3-dicarboxylate (1.0 eq.) inTHF at a concentration of 0.08 M was cooled at 0° C. and then LiCl (2.3eq.) and sodium borohydride (2.3 eq.) were added. After stirring for 20hours as the reaction warmed to rt, the pH was adjusted with 1M citricacid to pH 4-5. After removal of the volatiles in vacuo, the product wasextracted in dichloromethane, washed with H₂O and brine, dried overMgSO₄. Upon filtering and removal of the volatiles in vacuo,cis(±)-benzyl3-(tert-butoxycarbonylamino)-5-(hydroxymethyl)piperidine-1-carboxylatewas obtained as a white foamy solid. LCMS (m/z): 265.0 (MH-Boc⁺); LCR_(t)=3.37 min.

Synthesis of cis(±)-benzyl3-(tert-butoxycarbonylamino)-5-((tert-butyldimethylsilyloxy)methyl)piperidine-1-carboxylate

A solution of cis(±)-benzyl3-(tert-butoxycarbonylamino)-5-(hydroxymethyl)piperidine-1-carboxylate(1.0 eq.), imidazole (1.1 eq.), tert-butyldimethylsilylchloride (1.1eq.) and dimethylaminopyridine (0.1 eq.) in dichloromethane at aconcentration of 0.1 M was stirred for 18 hours at which time thevolatiles were removed in vacuo. Direct purification of the crudematerial by column chromatography (20% ethyl acetate/hexanes) yieldedcis(±)-benzyl3-(tert-butoxycarbonylamino)-5-((tert-butyldimethylsilyloxy)methyl)piperidine-1-carboxylate.LCMS (m/z): 379.0 (MH-Boc⁺); LC R_(t)=5.95 min.

Synthesis of cis(±)-tert-butyl5-((tert-butyldimethylsilyloxy)methyl)piperidin-3-ylcarbamate

Method 2 was followed to deprotect cis(±)-benzyl3-(tert-butoxy-carbonylamino)-5-((tert-butyldimethylsilyloxy)methyl)piperidine-1-carboxylateyielding cis(±)-tert-butyl5-((tert-butyldimethylsilyloxy)methyl)piperidin-3-ylcarbamate. LCMS(m/z): 344.1 (MH⁺).

Synthesis of cis(±)-tert-butyl5-((tert-butyldimethylsilyloxy)methyl)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

Method 1 was followed using cis(±)-tert-butyl5-((tert-butyldimethylsilyloxy)methyl)piperidin-3-ylcarbamate and4-chloro-3-nitropyridine yielding cis(±)-tert-butyl5-((tert-butyldimethylsilyloxy)methyl)-1-(3-nitropyridin-4-yl)-piperidin-3-ylcarbamate.LCMS (m/z): 467.0 (MH⁺); LC R_(t)=4.02 min.

Synthesis of cis(±)-tert-butyl1-(3-aminopyridin-4-yl)-5-((tert-butyldimethylsilyloxy)methyl)piperidin-3-ylcarbamate

Following Method 2, cis(±)-tert-butyl5-((tert-butyldimethylsilyloxy)methyl)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamatewas reduced yielding cis(±)-tert-butyl1-(3-aminopyridin-4-yl)-5-((tert-butyldimethylsilyloxy)methyl)piperidin-3-ylcarbamate.LCMS (m/z): 437.2 (MH⁺); LC R_(t)=3.86 min.

Synthesis of cis(±)-benzyl3-(tert-butoxycarbonylamino)-5-(fluoromethyl)piperidine-1-carboxylate

A solution of cis(±)-benzyl3-(tert-butoxycarbonylamino)-5-(hydroxymethyl)piperidine-1-carboxylate(1 eq.), perfluorobutanesulfonylfluoride (2 eq.), triethylamine-HF (4eq.) and triethylamine (6 eq.) in tetrahydrofuran at a concentration of0.16 M was stirred for 36 hours. Upon dilution with ethyl acetate (50×)the solution was washed with 1N HCl, NaHCO_(3(sat.)) and brine, wasdried over MgSO₄, filtered, concentrated and purified by columnchromatography (25-40% ethyl acetate/hexanes) to yield cis(±)-benzyl3-(tert-butoxycarbonylamino)-5-(fluoromethyl)piperidine-1-carboxylate(45% yield). LCMS (m/z): 267.1 (MH⁺); LC R_(t)=4.23 min.

Synthesis of cis(±)-tert-butyl 5-(fluoromethyl)piperidin-3-ylcarbamate

Method 2 was followed to deprotect cis(±)-benzyl3-(tert-butoxycarbonylamino)-5-(fluoromethyl)piperidine-1-carboxylateyielding cis(±)-tert-butyl 5-(fluoromethyl)piperidin-3-ylcarbamate. LCMS(m/z): 233.1 (MH⁺).

Synthesis of cis(±)-tert-butyl5-(fluoromethyl)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

Method 1 of example 1 was followed using cis(±)-tert-butyl5-(fluoromethyl)piperidin-3-ylcarbamate and 4-chloro-3-nitropyridineyielding cis(±)-tert-butyl5-(fluoromethyl)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate. LCMS(m/z): 355.1 (MH⁺); LC R_(t)=2.41 min.

Synthesis of cis(±)-tert-butyl1-(3-aminopyridin-4-yl)-5-(fluoromethyl)piperidin-3-ylcarbamate

Following Method 2, cis(±)-tert-butyl5-(fluoromethyl)-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate wasreduced yielding cis(±)-tert-butyl1-(3-aminopyridin-4-yl)-5-(fluoromethyl)piperidin-3-ylcarbamate. LCMS(m/z): 325.1 (MH⁺); LC R_(t)=2.27 min.

Synthesis of (3R,4R)-benzyl3-(tert-butoxycarbonylamino)-4-(methylsulfonyloxy)piperidine-1-carboxylate

To a solution of (3R,4R)-benzyl3-(tert-butoxycarbonylamino)-4-hydroxypiperidine-1-carboxylate indichloromethane (0.13 M) was added triethylamine (1.5 equiv.) followedby methanesulfonyl chloride (1.3 equiv.). The reaction was allowed tostir at room temperature for 15 h. The solution was then quenched withsaturated NaHCO₃, extracted with dichloromethane, dried with sodiumsulfate, and concentrated to give the crude (3R,4R)-benzyl3-(tert-butoxycarbonylamino)-4-(methylsulfonyloxy)piperidine-1-carboxylatein >95% yield. LCMS (m/z): 428.9/328.9 (MH⁺), R_(t)=3.81 min.

Synthesis of (3aR,7aS)-benzyl2-oxohexahydrooxazolo[4,5-c]pyridine-5(6H)-carboxylate

A solution of (3R,4R)-benzyl3-(tert-butoxycarbonylamino)-4-(methylsulfonyloxy)piperidine-1-carboxylatein pyridine (0.16 M) was heated to 120° C. in the microwave for 10minutes. The solution was then concentrated to almost dryness and theforming solid was filtered to give the desired product. The filtrate wasfurther purified via silica gel column chromatography eluting with ethylacetate (100%) to yield (3aR,7aS)-benzyl2-oxohexahydrooxazolo[4,5-c]pyridine-5(6H)-carboxylate in 75% combinedyield. LCMS (m/z): 277.1 (MH⁺), R_(t)=2.327 min.

Synthesis of (3aR,7aS)-5-benzyl 3-tert-butyl2-oxotetrahydrooxazolo[4,5-c]pyridine-3,5(2H,6H)-dicarboxylate

To a solution of (3aR,7aS)-benzyl2-oxohexahydrooxazolo[4,5-c]pyridine-5(6H)-carboxylate (1.0 equiv.) indichloromethane (0.09 M) was added BOC₂O (1.1 equiv.), triethylamine(1.1 equiv.), and a catalytic amount of DMAP. The reaction was stirredat room temperature for one hour at which point it was concentratedunder vacuo and filtered through a plug of silica gel eluting withethylacetate. The product was dried under vacuo to yield(3aR,7aS)-5-benzyl 3-tert-butyl2-oxotetrahydrooxazolo[4,5-c]pyridine-3,5(2H,6H)-dicarboxylate as awhite solid in 75% yield. LCMS (m/z): 277.2 (MH⁺), R_(t)=3.43 min.

Synthesis of (3aR,7aS)-tert-butyl5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate

To a solution of (3aR,7aS)-5-benzyl 3-tert-butyl2-oxotetrahydrooxazolo[4,5-c]pyridine-3,5(2H,6H)-dicarboxylate in amixture of EtOH and EtOAc (1:1, 0.07 M) was added Pd/C (10% by weight)and the reaction was stirred under a hydrogen balloon for 15 h. Thesolution was then filtered through a pad of Celite and the filtrate wasconcentrated to dryness to give a clear oil. To a solution of(3aR,7aS)-tert-butyl2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate in i-PrOH (0.12M) was added 4-chloro-3-nitropyridine (1.2 equiv.) and DIEA (4.0 equiv.)The reaction was heated to 75° C. for 2 h, then cooled to roomtemperature and concentrated under vacuo. The crude mixture was dilutedwith EtOAc, water was added, the organic layer was extracted, washedwith brine, dried with Na₂SO₄, and concentrated. The crude was purifiedvia silica gel column chromatography eluting with EtOAc (100%) to yield(3aR,7aS)-tert-butyl5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylateas a yellow foam in 89% yield). LCMS (m/z): 365.1 (MH⁺), R_(t)=1.79 min.

Synthesis of (3aR,7aS)-tert-butyl5-(3-aminopyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate

To a solution of (3aR,7aS)-tert-butyl5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylatein EtOH and EtOAc (1:1, 0.15 M) was added Pd/C (10% by weight) and thereaction was stirred under a hydrogen balloon for 15 h. The solution wasfiltered through a pad of Celite, and the filtrate was concentrated toyield (3aR,7aS)-tert-butyl5-(3-aminopyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylateas a clear oil in >95% yield. LCMS (m/z): 335.0 (MH⁺), R_(t)=1.68 min.

Synthesis of benzyl 3-azido-4-hydroxypiperidine-1-carboxylate and benzyl4-azido-3-hydroxypiperidine-1-carboxylate

To a solution of benzyl 7-oxa-3-azabicyclo[4.1.0]heptane-3-carboxylate(1.0 equiv.) in MeOH and water (0.17M) was added sodium azide (2.0equiv.) and ammonium chloride (1.0 equiv.). The reaction was stirred at65° C. in an oil bath for 7 h, then concentrated to remove the methanol.Ethyl acetate was added, the organic phase was separated, dried withNa₂SO₄, and concentrated under vacuo to give benzyl3-azido-4-hydroxypiperidine-1-carboxylate and benzyl4-azido-3-hydroxypiperidine-1-carboxylate as a clear oil in >95% yield.LCMS (m/z): 276.9 (MH⁺), R_(t)=2.98 min.

Synthesis of benzyl 3,7-diazabicyclo[4.1.0]heptane-3-carboxylate

To a solution of benzyl 3-azido-4-hydroxypiperidine-1-carboxylate andbenzyl 4-azido-3-hydroxypiperidine-1-carboxylate (1.0 equiv.) in dioxane(0.14M) was added PPh₃ (2.0 equiv.) and the reaction was heated toreflux for 1 h. The solution was then concentrated under vacuo andpurified via silica gel column chromatography eluting with DCM, 10% MeOHand 1% Et₃N to give benzyl 3,7-diazabicyclo[4.1.0]heptane-3-carboxylateas a clear oil in 25% yield. LCMS (m/z): 233.0 (MH⁺), R_(t)=1.94 min.

Synthesis of benzyl7-(diethoxyphosphoryl)-3,7-diazabicyclo[4.1.0]heptane-3-carboxylate

To a solution of benzyl 3,7-diazabicyclo[4.1.0]heptane-3-carboxylate(1.0 equiv.) in DCM (0.26M) was added diethyl phosphorochloridate (1.3equiv.) and triethyl amine (1.5 equiv.). The reaction was stirred for 24h, then concentrated to dryness. Added water and ethyl acetate, theorganics were extracted, dried with Na₂SO₄, and concentrated. The crudewas purified via silica gel column chromatography eluting with ethylacetate and hexanes (50% to 100% ethyl acetate) to yield benzyl7-(diethoxyphosphoryl)-3,7-diazabicyclo[4.1.0]heptane-3-carboxylate as aclear oil in 21% yield. LCMS (m/z): 369.0 (MH⁺).

Synthesis of benzyl3-(diethoxyphosphorylamino)-4-methylpiperidine-1-carboxylate

To a suspension of CuI (0.3 equiv.) in anhydrous THF (0.1M) was addedmethyl magnesium bromide (3M solution in Et₂O, 10 equiv.) at −40° C. Thereaction was stirred for 30 min, followed by addition of benzyl7-(diethoxyphosphoryl)-3,7-diazabicyclo[4.1.0]heptane-3-carboxylate (1.0equiv.) in THF (0.1M) at −40° C. Allowed the reaction to warm to 10° C.over 5 h, then quenched with water and extracted with ethyl acetate. Theorganic phase was concentrated under vacuo and purified via silica gelcolumn chromatography eluting with ethyl acetate and hexanes (50% to100% ethyl acetate) to yield benzyl3-(diethoxyphosphorylamino)-4-methylpiperidine-1-carboxylate in 35%yield. LCMS (m/z): 385.0 (MH⁺), R_(t)=3.38 min.

Synthesis of diethyl 4-methylpiperidin-3-ylphosphoramidate

To a solution of benzyl3-(diethoxyphosphorylamino)-4-methylpiperidine-1-carboxylate (1.0equiv.) in degassed MeOH was added Pd/C (10% by weight) and the reactionwas stirred under a hydrogen atmosphere for 1 h. The solution wasfiltered, then concentrated to yield diethyl4-methylpiperidin-3-ylphosphoramidate in 83% yield. LCMS (m/z): 251.1(MH⁺).

Synthesis of diethyl4-methyl-1-(3-nitropyridin-4-yl)piperidin-3-ylphosphoramidate

To a solution of diethyl 4-methylpiperidin-3-ylphosphoramidate (1.0equiv.) in isopropyl alcohol was added 4-chloro-3-nitropyridine (2.0equiv.) and DIEA (1.1 equiv.). The reaction was heated to 70° C. for 18h, then quenched with water and extracted with ethyl acetate. Theorganics were dried and concentrated under vacuo. The crude was purifiedvia ISCO (ethyl acetate and hexanes) to yield diethyl4-methyl-1-(3-nitropyridin-4-yl)piperidin-3-ylphosphoramidate in 52%yield. LCMS (m/z): 373.0 (MH⁺), R_(t)=1.93 min.

Synthesis of diethyl1-(3-aminopyridin-4-yl)-4-methylpiperidin-3-ylphosphoramidate

To a solution of diethyl4-methyl-1-(3-nitropyridin-4-yl)piperidin-3-ylphosphoramidate (1.0equiv.) in degassed EtOAc (0.1M) was added Pd/C (10% by weight) and thereaction was stirred under a hydrogen atmosphere for 18 h. Filtered andconcentrated the filtrate to yield diethyl1-(3-aminopyridin-4-yl)-4-methylpiperidin-3-ylphosphoramidate in 86%yield. LCMS (m/z): 343.0 (MH⁺), R_(t)=1.85 min.

Synthesis of benzyl4-chloro-3-(diethoxyphosphorylamino)piperidine-1-carboxylate

To a solution of benzyl7-(diethoxyphosphoryl)-3,7-diazabicyclo[4.1.0]heptane-3-carboxylate (1.0equiv.) and triethyl amine hydrochloride (4 equiv.) in DCM (0.1M) wasadded BF₃.OEt₂ (2.0 equiv.) and the reaction was allowed to stir at roomtemperature for 2 h under a nitrogen atmosphere. The solution was thenquenched with water and extracted with DCM. The crude product waspurified via silica gel column chromatography (ISCO eluting with EtOAcand Hexanes 50% to 100% EtOAc) to yield benzyl4-chloro-3-(diethoxyphosphorylamino)piperidine-1-carboxylate in 89%yield. LCMS (m/z): 405.1 (MH⁺), R_(t)=2.73 min.

Synthesis of diethyl 4-chloropiperidin-3-ylphosphoramidate

To a solution of benzyl4-chloro-3-(diethoxyphosphorylamino)piperidine-1-carboxylate (1.0equiv.) in degassed MeOH was added Pd/C (10% by weight) and the reactionwas stirred under a hydrogen atmosphere for 1 h. The solution wasfiltered, then concentrated to yield diethyl4-chloropiperidin-3-ylphosphoramidate in 92% yield. LCMS (m/z): 271.0(MH⁺).

Synthesis of diethyl4-chloro-1-(3-nitropyridin-4-yl)piperidin-3-ylphosphoramidate

To a solution of diethyl 4-chloropiperidin-3-ylphosphoramidate (1.0equiv.) in isopropyl alcohol (0.1M) was added 4-chloro-3-nitropyridine(2.0 equiv.) and DIEA (1.1 equiv.). The reaction was heated to 70° C.for 18 h, then quenched with water and extracted with ethyl acetate. Theorganics were dried and concentrated under vacuo. The crude was purifiedvia ISCO (ethyl acetate and hexanes then 10% methanol in DCM) to yielddiethyl 4-chloro-1-(3-nitropyridin-4-yl)piperidin-3-ylphosphoramidate in69% yield. LCMS (m/z): 393.1 (MH⁺), R_(t)=2.01 min.

Synthesis of diethyl1-(3-aminopyridin-4-yl)-4-chloropiperidin-3-ylphosphoramidate

To a solution of diethyl4-chloro-1-(3-nitropyridin-4-yl)piperidin-3-ylphosphoramidate (1.0equiv.) in degassed EtOAc (0.1M) was added Pd/C (10% by weight) and thereaction was stirred under a hydrogen atmosphere for 18 h. Filtered andconcentrated the filtrate to yield diethyl1-(3-aminopyridin-4-yl)-4-chloropiperidin-3-ylphosphoramidate in 83%yield. LCMS (m/z): 363.1 (MH⁺), R_(f)=1.89 min.

Synthesis of 3-oxocyclohex-1-enyl trifluoromethanesulfonate

To a solution of cyclohexane-1,3-dione (1 equiv) in DCM (0.4M) was addedNa₂CO₃ (1.0 equiv.) and cooled to 0° C. Added Tf₂O (1.0 equiv.) in DCM(5M) dropwise over 1 hr at room temperature under a nitrogen atmosphere.Upon addition, the reaction was stirred for 2 hr (dark red solution).The solution was filtered and to the filtrate was added saturated NaHCO₃(carefully), then extracted the organics, dried with brine, then Na₂SO₄,and concentrated. The crude was purified via SiO₂ column chromatographyeluting with DCM and hexanes (1:1) or alternatively via a neutralalumina plug eluting with DCM to afford 3-oxocyclohex-1-enyltrifluoromethanesulfonate in 30% or 67% yield respectively. The triflatedecomposes upon storage and should be used immediately for the nextreaction. LC/MS=244.9/286.0 (M+H and M+CH₃CN); Rt=0.88 min.

Synthesis of3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone

To a solution of 3-oxocyclohex-1-enyl trifluoromethanesulfonate (1.0equiv.) in degassed dioxane (0.3M) was added bis(pinacolato)diboron (2.0equiv.), KOAc (3.0 equiv.), and Pd(dppf)Cl₂-DCM (0.05 equiv.). Thereaction was heated to 80° C. for 2 h, then filtered. The dioxanesolution was used for the next step without further purification.LC/MS=140.9 (M+H of boronic acid).

Synthesis of 3-(3-nitropyridin-4-yl)cyclohex-2-enone

To a solution of3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone (1.0equiv.) in degassed dioxane and 2M Na₂CO₃ was added4-chloro-3-nitropyridine (1.2 equiv.) and Pd(PPh₃)₄ (0.05 equiv.). Thereaction was heated in an oil bath to 120° C. for 30 min. (reaction canalso be carried out in the microwave for 10 min at 120° C.). Cooled toroom temperature, then diluted with EtOAc, added H₂O—dark solution, lotsof emulsions. Filtered to get rid of the solids, then extracted theorganic phase, dried with Na₂SO₄, and concentrated. The crude waspurified via silica gel chromatography to yield3-(3-nitropyridin-4-yl)cyclohex-2-enone (64%, 2 steps). LC/MS=219 (M+H),LC=2.29 min.

Synthesis of 3-(3-nitropyridin-4-yl)cyclohex-2-enol

To a solution of 3-(3-nitropyridin-4-yl)cyclohex-2-enone (1.0 equiv.)was added EtOH (1.1 M) and CeCl₃-7H₂O (1.3 equiv.). The reaction wascooled to 0° C., then NaBH₄ (1.3 equiv.) was added in portions. Stirredfor 2 h at 0° C., then quenched by adding water, concentrated to removethe EtOH, added EtOAc, extracted the organics, dried with brine, thenNa₂SO₄, and concentrated to yield 3-(3-nitropyridin-4-yl)cyclohex-2-enol(99%). LC/MS=221.1 (M+H), LC=2.24 min.

Synthesis of2-(3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione

To a homogeneous solution of 3-(3-nitropyridin-4-yl)cyclohex-2-enol (1.0eq), triphenyl phosphine (1.5 eq), and phthalimide (1.5 eq) in THF (0.2M) cooled to 0° C., ditertbutyl azodicarboxylate (1.5 eq) in THF wasadded to the solution. The mixture was stirred at 0° C. for 2 hours. Thereaction was concentrated in vacco. The residue was purified by column(5% methanol in 1:1 ethyl acetate and hexanes) to give a solid, whichwas further triturated with DCM and hexanes to yield pure product, plusfiltrate. Further purification of the filtrate yielded more pureproduct. The total yield of2-(3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione is 58%.LC/MS (m/z): MH⁺=350.2, Rt=0.96. HPLC Rt=3.73.

Synthesis of 2-(3-(3-aminopyridin-4-yl)cyclohexyl)isoindoline-1,3-dione

A solution of2-(3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione (1 eq)in Acetic Acid (0.1 M) was purged with nitrogen for 10 min. Then 10%Pd/C (0.15 eq) was added. The reaction mixture was stirred at roomtemperature for four days under an atmosphere of hydrogen. Solids wereremoved by filtration over celite, then rinsed with EtOAc and MeOH. Thefiltrate was concentrated, diluted with EtOAc and washed 2× with sat.aq. 2M Na₂CO₃. Organic layer was dried with MgSO₄, filtered, andconcentrated. Triturated from EtOAc/hexanes to give2-(3-(3-aminopyrin-4-yl)cyclohexyl)isoindoline-1,3-dione in 77% yield.LC/MS (m/z): MH⁺=322.2, Rt=0.64. HPLC Rt=2.43 min.

Synthesis of 5,5-dimethyl-3-oxocyclohex-1-enyl trifluoromethanesulfonate

In a 3-neck round bottom flask, 5,5-dimethylcyclohexane-1,3-dione (1.0eq) was dissolved in DCM (0.2 M). Sodium carbonate (1.1 equiv.) wasadded and the mixture cooled with magnetic stirring on an ice/salt/waterbath to ˜−5° C. under N₂. Triflic anhydride (1.05 equiv.) diluted in DCMwas added drop wise via addition funnel over 90 minutes. Upon completionof addition, the reaction was stirred at ˜0° C. for 1 h. From LCMS and¹H NMR, there was still starting material left. Additional sodiumcarbonate (0.51 equiv.) and triflic anhydride (0.50 equiv.) were added.After 2 hours, the mixture was filtered through a coarse frit glassfunnel (the cake was washed with DCM), transferred to an Erlenmeyerflask, quenched by careful addition of saturated aqueous sodiumbicarbonate with vigorous stirring until pH=7, transferred to aseparatory funnel and the layers separated. The organic layer was washedwith brine, dried over MgSO₄, filtered, concentrated to give5,5-dimethyl-3-oxocyclohex-1-enyl trifluoromethanesulfonate, which wasused to next step without further purification. LC/MS (m/z): MH⁺=273.1,Rt=1.03 min.

Synthesis of5,5-dimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone

All of reagents 5,5-dimethyl-3-oxocyclohex-1-enyltrifluoromethanesulfonate (1.0 eq), potassium acetate (3.0 eq), andbis(pinacolato)diboron (2.0 eq) were added to 1,4-dioxane (0.2 M) in around bottom flask and degassed by bubbling N₂ through the mixture for10 min. PdCl₂(dppf)-DCM adduct (0.03 eq) was added and the reactionheated to 80° C. fitted with a reflux condenser on an oil bath under N₂overnight. The mixture was cooled to room temperature, filtered througha coarse frit glass funnel, the cake rinsed with 1,4-dioxane to give the5,5-dimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enonein 1,4-dioxane which was used to next step without further purification.LC/MS (m/z): MH⁺ (boronic acid)=169.1, Rt=0.50 min.

Synthesis of 5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone

The boronate ester5,5-dimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone(1.0 eq) was dissolved in 1,4-dioxane in a round bottom flask wasdegassed by bubbling N₂ through the solution for 30 minutes.4-chloro-3-nitro-pyridine (1.3 eq) and 2M(aq) sodium carbonate (2.0 eq)were added and N₂ was bubbled through for 10 minutes and thenPdCl₂(dppf)-DCM (0.05 eq) was added. The reaction mixture was stirred at110° C. for 2 hr. The mixture was added EtOAc and water. The resultingmixture was filtered through celite, the cake was washed with EtOAc. Theorganic layer was separated. The aqueous was extracted with EtOAc. Thecombined organic layers were washed with brine, dried over MgSO₄,concentrated. The residue was purified by silica gel chromatography(eluted with EtOAc:Hexanes=1:10 to 2:1) to give5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone (46.7% for threesteps). LC/MS (m/z): MH⁺=247.2, Rt=0.79 min.

Synthesis of 5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol

To a solution of 5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone(1.0 eq), and CeCl₃-7H₂O (1.2 eq) in MeOH (0.2 M) was added NaBH₄ (1.0eq) at 0° C. The solution was stirred for 1 hour, and then quenched by 5mL of water. The volatiles were removed in vacuum and the residue waspartitioned between EtOAc and H₂O. The organic layer was separated andwashed with brine. The combined aqueous was back extracted with EtOAcand the organic was washed with brine. The combined organics were driedover MgSO₄, filtered and concentrated. The residue was purified bycolumn (5% methanol in 1:1 ethyl acetate and hexanes) to give5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (74%). LC/MS (m/z):MH⁺=249.2, Rt=0.76 min.

Synthesis of2-(5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione

To a homogeneous solution of5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (1.0 eq), triphenylphosphine (1.5 eq), and phthalimide (1.5 eq) in THF (0.2 M) cooled to 0°C., ditertbutyl azodicarboxylate (1.5 eq) in THF was added to thesolution. The mixture was stirred at 0° C. for 2 hours. The reaction wasconcentrated in vacco. The residue was purified by column (5% methanolin 1:1 ethyl acetate and hexanes) to give2-(5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione(99%). LC/MS (m/z): MH⁺=378.2, Rt=1.10 min.

Synthesis of2-(3-(3-aminopyridin-4-yl)-5,5-dimethylcyclohex-2-enyl)isoindoline-1,3-dione

A solution of2-(5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione(1 eq) in Acetic Acid (0.1 M) was purged with nitrogen for 10 min. Then10% Pd/C (0.10 eq) was added. The reaction mixture was stirred at roomtemperature overnight an atmosphere of hydrogen. Solids were removed byfiltration over celite, then rinsed with EtOAc and MeOH. The filtratewas concentrated, diluted with EtOAc and washed 2× with sat. aq. 2MNa₂CO₃. Organic layer was dried with MgSO₄, filtered, and concentrated.The residue was purified by column (5% methanol in 1:1 ethyl acetate andhexanes) to give2-(3-(3-aminopyridin-4-yl)-5,5-dimethylcyclohex-2-enyl)isoindoline-1,3-dione(89%). LC/MS (m/z): MH⁺=348.3, Rt=0.79 min.

Synthesis of2-(5-(3-aminopyridin-4-yl)-3,3-dimethylcyclohexyl)isoindoline-1,3-dione

A solution of2-(3-(3-aminopyridin-4-yl)-5,5-dimethylcyclohex-2-enyl)isoindoline-1,3-dione(1.0 eq) in acetic acid (0.1 M) was purged with nitrogen for 10 min.Then 10% Pd/C (0.1 eq) was added. The reaction mixture was stirred at45° C., 300 psi hydrogen atmosphere in a steel bomb overnight and at 65°C., 300 psi for 5 hours. Solids were removed by filtration over celite,then rinsed with EtOAc and MeOH. The filtrate was concentrated, dilutedwith EtOAc and washed 2× with sat. aq. 2M Na₂CO₃. Organic layer wasdried with MgSO₄, filtered, and concentrated. The residue was purifiedby column (5% methanol in 1:1 ethyl acetate and hexanes) to give2-(5-(3-aminopyridin-4-yl)-3,3-dimethylcyclohexyl)isoindoline-1,3-dione(53%). LC/MS (m/z): MH⁺=350.3, Rt=0.78 min. The enantiomerically pure2-((1R,5R)-5-(3-aminopyridin-4-yl)-3,3-dimethylcyclohexyl)isoindoline-1,3-dioneand2-((1S,5S)-5-(3-aminopyridin-4-yl)-3,3-dimethylcyclohexyl)isoindoline-1,3-dionewere resolved by chiral HPLC (For analysis R_(t)=7.53 min and 13.11 minrespectively; hexanes:ethanol=80:20 (v:v), Chiralcel OJ-H 100×4.6 mm at1 mL/min. For preparative separation, hexanes:ethanol=80:20 (v:v),Chiralcel OJ-H 250×20 mm at 20 mL/min ). ¹H NMR (CDCl₃): δ 8.04 (s, 1H),8.00 (d, 1H), 7.82 (m, 2H), 7.71 (m, 2H), 7.06 (d, 1H), 4.54 (m, 1H),3.71 (m, 2H), 2.89 (m, 1H), 2.23-2.44 (m, 2H), 1.90 (m, 1H), 1.20-1.60(m, 3H), 1.18 (s, 3H), 1.07 (s, 3H).

Synthesis of 5-methyl-3-oxocyclohex-1-enyltrifluoromethanesulfonate

To a solution of 5-methylcyclohexane-1,3-dione (1 equiv) in DCM (0.4M)was added Na₂CO₃ (1.0 equiv.) and cooled to 0° C. Added Tf₂O (1.0equiv.) in DCM (5M) dropwise over 1 hr at 0° C. under a nitrogenatmosphere. Upon addition, the reaction was stirred for 2 hr at roomtemperature (dark red solution). The solution was filtered and to thefiltrate was added saturated NaHCO₃ (carefully), then extracted theorganics, dried with brine, then Na₂SO₄, and concentrated. The crude waspurified via SiO₂ column chromatography eluting with DCM and hexanes(1:1) or alternatively via a neutral alumina plug eluting with DCM toafford 5-methyl-3-oxocyclohex-1-enyl trifluoromethanesulfonate in 30% or67% yield respectively. The triflate decomposes upon storage and shouldbe used immediately for the next reaction. LC/MS=259.1/300.1 (M+H andM+CH₃CN); Rt=0.94 min, LC=3.84 min.

Synthesis of5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone

To a solution of 5-methyl-3-oxocyclohex-1-enyl trifluoromethanesulfonate(1.0 equiv.) in degassed dioxane (0.3M) was added bis(pinacolato)diboron(2.0 equiv.), KOAc (3.0 equiv.), and Pd(dppf)Cl₂-DCM (0.05 equiv.). Thereaction was heated to 80° C. for 10 h, then filtered. The dioxanesolution was used for the next step without further purification.LC/MS=155.1 (M+H of boronic acid); Rt=0.41 min, LC=1.37 min.

Synthesis of 5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone

To a solution of5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone(1.0 equiv.) in degassed dioxane and 2M Na₂CO₃ was added4-chloro-3-nitropyridine (1.2 equiv.) and Pd(PPh₃)₄ (0.05 equiv.). Thereaction was heated in an oil bath to 120° C. for 2 h. (reaction canalso be carried out in the microwave for 10 min at 120° C.). Cooled toroom temperature, then diluted with EtOAc, added H₂O—dark solution, lotsof emulsions. Filtered to get rid of the solids, then extracted theorganic phase, dried with Na₂SO₄, and concentrated. The crude waspurified via silica gel chromatography to yield5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone. LC/MS=233.2 (M+H);Rt=0.69 min, LC=2.70 min.

Synthesis of cis-(±)-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol

To a solution of 5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone (1.0equiv.) was added EtOH (1.1 M) and CeCl₃-7H₂O (1.3 equiv.). The reactionwas cooled to 0° C., then NaBH₄ (1.3 equiv.) was added in portions.Stirred for 2 h at 0° C., then quenched by adding water, concentrated toremove the EtOH, added EtOAc, extracted the organics, dried with brine,then Na₂SO₄, and concentrated to yield5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (91%). LC/MS=235.2(M+H), LC=2.62 min.

Synthesis ofcis-(±)-4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridine

To a solution of 5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (1.0equiv.) in DMF (0.5 M) was added imidazole (4.0 equiv.) and TBDSMCl (2.5equiv.). After stirring for 18 hours the solution was portioned betweenEtOAc and H₂O and separated. After washing further with H₂O (3×) andNaCl_((sat.),) drying over MgSO4, filtering and removal of solvents,4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridinewas obtained (85%). LC/MS=349.2 (M+H), LC=5.99 min.

Synthesis ofcis-(±)-4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine

To a solution ofcis-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridine(1.0 equiv.) in methanol, at a concentration of 0.1 M, was added 10%palladium on carbon (0.1 eq.). The resultant heterogeneous solution wasput under an atmosphere of hydrogen and was stirred for 15 hours. Atthis time the mixture was filtered through a pad of celite eluting withmethanol. The volatiles were removed in vacuo yielding allcis-4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine(90%). LCMS (m/z): 321.3 (MH⁺); LC R_(t)=3.85 min.

Synthesis of cis(±)benzyl4-3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-ylcarbamate

To a solution ofcis-(±)-4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-aminein dichloromethane at a concentration of 0.5M was added benzyl2,5-dioxopyrrolidin-1-yl carbonate (1.1 equiv.) and DMAP (0.05 equiv.).After stirring for 16 hours at rt, additional benzyl2,5-dioxopyrrolidin-1-yl carbonate (0.55 equiv.) and DMAP (0.03 equiv.)were added. After stirring for an additional 24 hours at rt, additionalbenzyl 2,5-dioxopyrrolidin-1-yl carbonate (0.1 equiv.) and DMAP (0.03equiv.) were added. After stirring for 18 more hours the solution waspartitioned between EtOAc and Na₂CO_(3(sat.)) and separated. Uponfurther washing with Na₂CO_(3(sat.)) (2×) and NaCl_((sat.),) drying overMgSO₄, filtering and removal of solvents, cis(±)benzyl4-3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-ylcarbamatewas obtained. The crude material was used as is. LC/MS=455.3 (M+H),LC=4.39 min.

Synthesis of cis-(±)benzyl4-(3-hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamate

A solution of cis(±)benzyl4-3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-ylcarbamatein 1:2:1 6N HCl/THF/MeOH at a concentration of 0.1 M was stirred at rtfor 6 hours. The pH was than adjusted to pH=7 by addition of 6N NaOH andthe volatiles were removed in vacuo. The aqueous layer was extractedwith EtOAc and the organic was washed with NaCl_((sat.).) dried overMgSO₄, filtered and upon removal of the volatiles in vacuo,cis-(±)benzyl 4-(3-hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamate wasobtained. The crude material was used as is. LC/MS=341.2 (M+H), LC=2.38min.

Synthesis of cis(±)-benzyl4-(3-methyl-5-oxocyclohexyl)pyridin-3-ylcarbamate

To a 0° C. solution of cis-(±)-benzyl4-(3-hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamate in wet CH₂Cl₂ at aconcentration of 0.16 M was added Dess-Martin Periodinane (1.5 equiv.)and the solution was stirred for 18 hours as it warmed to rt. Thesolution was partitioned between EtOAc and 1:1 10%Na₂S₂O₃/NaHCO_(3(sat.)) and separated. Upon further washing with 1:1 10%Na₂S₂O₃/NaHCO_(3(sat.)) (2×) and NaCl_((sat.),) drying over MgSO₄,filtering, removal of solvents and purification by silica gelchromatography (75-100% EtOAc/hexanes),cis-(±)-benzyl-4-(3-methyl-5-oxocyclohexyl)pyridin-3-ylcarbamate wasobtained as a white solid (53%, 5 steps). LC/MS=339.2 (M+H).

Synthesis of cis-(±)-benzyl4-(-3-(benzylamino)-5-methylcyclohexyl)pyridin-3-ylcarbamate

A solution ofcis-(±)-benzyl-4-(3-methyl-5-oxocyclohexyl)pyridin-3-ylcarbamate (1.0equiv) and benzylamine (3.0 equiv) in MeOH, at a concentration of 0.25M, was stirred at rt for 2 hours. Upon cooling in a −78° C. bath, LiBH₄(1.1 equiv, 2.0 M in THF) was added and the solution was allowed to warmto rt with stirring over 16 hours. The solution was partitioned betweenEtOAc and NaHCO_(3(sat.),) separated, washed further withNaHCO_(3(sat.)) and NaCl_((sat.),) dried over MgSO₄, filtered and theafter removal of volatiles in vacuo, cis-(±)-benzyl4-(-3-(benzylamino)-5-methylcyclohexyl)pyridin-3-ylcarbamate wasobtained as a 4:1 mixture of isomers, with the all cis as predominantLC/MS=430.3 (M+H), LC=0.62 min.

Synthesis ofcis(±)-tert-butyl(-3-(3-aminopyridin-4-yl)-5-methylcyclohexylcarbamate

To a solution of cis-(±)-benzyl4-(-3-(benzylamino)-5-methylcyclohexyl)pyridin-3-ylcarbamate was (1.0equiv.) in methanol, at a concentration of 0.07 M, was added 20%palladium hydroxide on carbon (0.2 eq.). The resultant heterogeneoussolution was put under an atmosphere of hydrogen and was stirred for 14hours. At this time the reaction was purged with Ar, Boc₂O (1.0 equiv.)was added and the solution was stirred for 8 hours. Additional Boc₂O(1.0 equiv.) was added and the solution was stirred for 16 more hours.At this time the mixture was filtered through a pad of celite elutingwith methanol. Upon removal of volatiles in vacuo, purification bysilical gel chromatography (2.5-2.5 MeOH/CH₂Cl₂ with 0.1% DIEA) andrecrystallization from 10% EtOAc/hexanes yieldedcis(±)-tert-butyl(-3-(3-aminopyridin-4-yl)-5-methylcyclohexylcarbamate(49%). LCMS (m/z): 306.3 (MH⁺), LC R_(t)=2.59 min. Pure enantiomerscould be obtained by chiral chromatography.

Synthesis of 4-(cyclohexa-1,3-dienyl)-3-nitropyridine

To a solution of 3-(3-nitropyridin-4-yl)cyclohex-2-enol (1.0 equiv.) wasadded dioxane (0.18M) and P-TSA (1.1 equiv.). The solution was heated to100° C. for 4 h. Cooled to room temperature, worked up with sat. NaHCO₃and ethyl acetate, the organic phase was dried with Na₂SO₄ andconcentrated. The crude was purified via silica gel columnchromatography eluting with 100% DCM to give4-(cyclohexa-1,3-dienyl)-3-nitropyridine as a yellow oil (27% yield).LCMS (m/z): 203.1 (MH⁺), LC R_(t)=3.53 min, H-NMR (CDCl₃): 9.02 (s, 1H),8.70 (d, J=5.3, 1H), 7.30 (d, J=5.3, 1H), 6.15-6.17 (m, 1H), 6.02-6.11(m, 2H), 2.35-2.38 (m, 4H).

Synthesis of tert-butyl6-hydroxy-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate

To a solution of 2-azido-4-(3-nitropyridin-4-yl)cyclohex-3-enol (1.0equiv.) in Pyridine and NH₄OH (8:1, 0.23 M) was added trimethylphosphine(3.0 equiv.) at room temperature. The mixture was stirred at roomtemperature for 3 hours. Solvents were removed. To the residue was addedethanol. Then ethanol was removed in vacuo to ensure removal of theammonia totally. The residue was dissolved in 1,4-Dioxane and sat. aq.sodium bicarbonate, and then Boc₂O (1.0 eq) in THF were added to themixture. The resulting mixture was stirred at room temperature for 2hours. The reaction mixture was diluted with ethyl acetate, and washedwith sat NaCl. The organic was dried with MgSO₄, filtered andconcentrated. The residue was purified by column (5% methanol in 1:1ethyl acetate and hexanes) to give tert-butyl6-hydroxy-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate (82%). LC/MS(m/z): MH⁺=336.0, Rt=0.71

Synthesis of(±)-4-(3-azido-4-(tert-butyldimethylsilyloxy)cyclohex-1-enyl)-3-nitropyridine

To a solution of (±)-2-azido-4-(3-nitropyridin-4-yl)cyclohex-3-enol (1.0equiv.) in DCM (0.15M) was added TBSCl (2.0 equiv.), imidazole (2.0equiv.) and DMAP (0.1 equiv.) at room temperature. After 18 h, water wasadded, the organics were dried with brine, then Na₂SO₄, andconcentrated. The crude material was loaded to silica gel and purifiedvia ISCO eluting with ethyl acetate and hexanes (20%). Obtained(±)-4-(3-azido-4-(tert-butyldimethylsilyloxy)cyclohex-1-enyl)-3-nitropyridineas a yellow oil in 60% yield. LCMS (m/z): 376.3 (MH⁺), LC R_(t)=5.848min.

Synthesis of (±)-tert-butyl6-(tert-butyldimethylsilyloxy)-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate

In a round-bottomed flask was added(±)-4-(3-azido-4-(tert-butyldimethylsilyloxy)cyclohex-1-enyl)-3-nitropyridine(1.0 equiv.) and pyridine (0.1M) to give a yellow solution. Ammoniumhydroxide (10:1 pyridine:ammonium hydroxide) was added followed by PMe₃(3.0 equiv.). The reaction turned dark brown after 10 min. Stirred atroom temperature for 1.5 h. Quenched by adding EtOH, and concentrated.Repeated 2 more times. To the crude was added sat. NaHCO₃ and dioxane(1:1, 0.1M). Boc₂O (1.0 equiv.) was added. Stirred for one hour at roomtemperature. Washed with H₂O and EtOAc, the organic phase was dried withMgSO₄, filtered and concentrated. The residue was purified via ISCO (5:1Hex/EtOAc). Collected the pure fractions and concentrated to give(+)-tert-butyl6-(tert-butyldimethylsilyloxy)-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamateas a foam. LCMS (m/z): 450.3 (MH⁺), LC R_(t)=5.83 min.

Synthesis of (±)-tert-butyl3-(3-aminopyridin-4-yl)-6-(tert-butyldimethylsilyloxy)cyclohex-2-enylcarbamate

To a solution of (±)-tert-butyl6-(tert-butyldimethylsilyloxy)-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate(1.0 equiv.) in AcOH (0.18 M) was added Fe (6.0 equiv.) and the reactionwas stirred for 20 h. Worked up by diluting the reaction with methanol,filtered, and concentrated the filtrate. To the crude was added ethylacetate and saturated NaHCO₃, the organics were dried with sodiumsulfate and concentrated to give (±)-tert-butyl3-(3-aminopyridin-4-yl)-6-(tert-butyldimethylsilyloxy)cyclohex-2-enylcarbamateas a yellow oil in 94% yield. LCMS (m/z): 420.3 (MH⁺), LC R_(t)=3.88min.

Synthesis of (±)-tert-butyl5-(3-aminopyridin4-yl)-2-(tert-butyldimethylsilyloxy)cyclohexylcarbamate

To a solution of (±)-tert-butyl3-(3-aminopyridin-4-yl)-6-(tert-butyldimethylsilyloxy)cyclohex-2-enylcarbamate(1.0 equiv.) in MeOH (0.1M) was added Pd/C (20% by wt) and the reactionwas stirred under a hydrogen balloon for 18 h. LC/MS of the reactionindicated mixture of diastereomers, the reaction was filtered, washedwith EtOAc and concentrated the filtrate. The crude material waspurified via prep-HPLC (in DMSO), and the pure fractions were combined,neutralized with solid NaHCO₃, extracted with ethyl acetate, washed withbrine, dried under Na₂SO₄, and concentrated to give product A (8% yield)and product B (51% yield).

Product A: LCMS (m/z): 422.4 (MH⁺), LC R_(t)=3.75 min.

Product B: LCMS (m/z): 422.4 (MH⁺), LC R_(t)=3.94 min.

Synthesis of2-(tert-butoxycarbonylamino)-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate

To a solution of tert-butyl6-hydroxy-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate (1.0 equiv.)and triethyl amine (1.5 equiv.) in CH₂Cl₂ (0.2 M) was addedmethanesulfonyl chloride (1.2 equiv.) at 0° C. The mixture was stirredfor 2 hours at that temperature. The reaction mixture was diluted withethyl acetate, and washed with sat NaCl. The organic was dried withMgSO₄, filtered and concentrated to give2-(tert-butoxycarbonylamino)-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate (85%), which was used in the next step without furtherpurification. LC/MS (m/z): MH⁺=414.0, Rt=0.82

Synthesis of(±)-5-(3-nitropyridin-4-yl)-3,3a,7,7a-tetrahydrobenzo[d]oxazol-2(6H)-one

The mixture of2-(tert-butoxycarbonylamino)-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate (1.0 equiv.) in pyridine (0.21 M) was stirred at 110°C. for 10 min in microwave. Pyridine was removed under reduced pressure.The residue was dissolved in ethyl acetate, and washed with sat NaCl.The organic was dried with MgSO₄, filtered and concentrated to give5-(3-nitropyridin-4-yl)-3,3a,7,7a-tetrahydrobenzo[d]oxazol-2(6H)-one(85%), which was used in the next step without further purification.LC/MS (m/z): MH⁺=262.1, Rt=0.49

Synthesis of (±)-tert-butyl5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate

To a solution of5-(3-nitropyridin-4-yl)-3,3a,7,7a-tetrahydrobenzo[d]oxazol-2(6H)-one(1.0 equiv.), TEA (1.8 equiv.), and catalytic amount DMAP in CH₂Cl₂(0.19 M) was added di-tert-butyl dicarbonate (1.2 eqiv) at roomtemperature. The reaction mixture was stirred for 1 hour. The reactionmixture was diluted with ethyl acetate (100 mL), and washed with satNaCl (30 mL). The organic was dried with MgSO₄, filtered andconcentrated. The residue was purified by column (5% methanol in 1:1ethyl acetate and hexanes) to give tert-butyl5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate(98%). LC/MS (m/z): MH⁺=306.0, Rt=0.75

Synthesis of (±)-tert-butyl5-(3-aminopyridin-4-yl)-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylate

To a solution of tert-butyl5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate(1.0 equiv.) in methanol and ethyl acetate (1;1, 0.1 M) was added Pd/C(10%). The resulting mixture was stirred under H₂ atmosphere for 6hours. The solid was removed by filtration. The filtrate wasconcentrated under reduced pressure to give tert-butyl5-(3-aminopyridin-4-yl)-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylate(87%), which was used in the next step without further purification.LC/MS (m/z): MH⁺=334.1, Rt=0.5 1.

Synthesis of (±)-4-(5-methylcyclohexa-1,3-dienyl)-3-nitropyridine

To a solution of 5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (1.0equiv.) in dioxane (0.1M) was added p-TSA (1.0 equiv.), and the reactionwas stirred at 100° C. for 3 h. The solution was cooled to roomtemperature, then passed through a pad of neutral alumina eluting withEtOAc to yield (±)-4-(5-methylcyclohexa-1,3-dienyl)-3-nitropyridine as ayellow oil in 68% yield. LC/MS=217.1 (M+H), LC=3.908 min.

Synthesis of (±)-6-bromo-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol

To a solution of 4-(5-methylcyclohexa-1,3-dienyl)-3-nitropyridine (1.0equiv.) in THF and water (1:1, 0.13M) was added NBS (1.5 equiv.) and thereaction was stirred at room temperature for 30 min. Upon completion,ethyl acetate and water were added to the reaction, the organic phasewas dried with brine, then sodium sulfate, filtered, and concentrated.The crude material was purified via silica gel column chromatographyeluting with ethyl acetate and hexanes (1:1) to give(±)-6-bromo-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol as a yellowoil in 80% yield. LC/MS=315.0/313.0 (M+H), LC=2.966 min.

Synthesis of (±)-2-azido-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol

To a solution of(±)-6-bromo-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (1.0 equiv.)in THF (0.1M) was added potassium tert-butoxide (1.5 equiv.). Thereaction turned from orange to black almost immediately. By TLC, theformation of product is clean in 30 min. Quenched by adding saturatedammonium chloride and ethyl acetate. The organic phase was dried withbrine, then sodium sulfate, filtered, and concentrated. The crudeproduct was dissolved in ethanol and water (3:1, 0.1M), and ammoniumchloride (2.0 equiv) and sodium azide (2.0 equiv.) were added. The darkorange reaction was stirred at room temperature overnight. Theconversion to product is clean as indicated by LC/MS. The reaction wasconcentrated to remove the ethanol, ethyl acetate and water were added,the organic phase was dried with sodium sulfate, filtered, andconcentrated. The crude material was purified via silica gel columnchromatography eluting with ethyl acetate and hexanes (1:1) to give(±)-2-azido-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol in 55%yield. LC/MS=276.0 (M+H), LC=2.803 min.

Synthesis of (±)-tert-butyl6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate

To a solution of(±)-2-azido-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol (1.0 equiv.)in pyridine and ammonium hydroxide (8:1, 0.08 M) was addedtrimethylphosphine (3.0 equiv.) and the brown solution was stirred atroom temperature for 2 h. Upon completion, EtOH was added and thesolution was concentrated in vacuo. More ethanol was added and thereaction was concentrated again. Dioxane and sat. NaHCO₃ (1:1, 0.08M)were added to the crude, followed by Boc₂O (1.0 equiv.). Stirred thereaction mixture at room temperature for 2 h, then added water and ethylacetate. The organic phase was dried with MgSO₄, and concentrated. Thecrude product was purified via silica gel column chromatography elutingwith ethyl acetate and hexanes (1:1) to afford (±)-tert-butyl6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate(59%). LC/MS=350.1 (M+H), Rt: 0.76 min.

Synthesis of(±)-2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylacetate

To a solution of (±)-tert-butyl6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate (1.0equiv.) in pyridine (0.1M) was added Ac2O (2.0 equiv.) and the reactionwas stirred at room temperature overnight. Upon completion, the reactionwas concentrated to dryness, then worked-up with ethyl acetate andwater. The organic phase was dried with brine, then sodium sulfate,filtered, and concentrated to give(±)-2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylacetate in 94% yield. LC/MS=392.2 (M+H), Rt=0.94 min.

Synthesis of(±)-4-(3-aminopyridin-4-yl)-2-(tert-butoxycarbonylamino)-6-methylcyclohexylacetate

To a degassed solution of(±)-2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylacetate (1.0 equiv.) in MeOH and EtOAc (1:1, 0.1M) was added Pd/C (0.1equiv.) and the reaction was stirred at room temperature under ahydrogen balloon for 3 days. Upon completion, the solution was filteredthrough a pad of Celite, the pad was washed with ethyl acetate and thefiltrate was concentrated. The crude material contained about 10% of theundesired isomer. The crude was dissolved in ethyl acetate (˜20%) andhexanes and heated until all dissolved. The solution was allowed to sitat room temperature for 2 days. The precipitate was then collected togive(±)-4-(3-aminopyridin-4-yl)-2-(tert-butoxycarbonylamino)-6-methylcyclohexylacetate as the pure product in 59% yield. LC/MS=364.3 (M+H), Rt=0.63min.

Synthesis of2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate

To a solution of tert-butyl6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate (1.0equiv.) in DCM (0.09 M) was added triethylamine (1.5 equiv.) and thereaction was cooled to 0° C. MsCl (1.2 equiv.) was added to the reactionand stirred for 3 h. Another 1.0 equiv. of MsCl was added to thereaction and stirred for another 2 h. Worked up the reaction by addingwater, the organic phase was dried with brine, sodium sulfate, andconcentrated. The crude product was purified via silica gel columnchromatography eluting with ethyl acetate and hexanes (1:1) to afford2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate as a white foam in 65% yield. LC/MS=428.2 (M+H), LC:3.542 min.

Synthesis of (±)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate

A solution of(±)-2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate (1.0 equiv.) in pyridine (0.2M) was heated in themicrowave at 110° C. for 10 min. The orange reaction was thenconcentrated under vacuo, the crude was dissolved in ethyl acetate andwater, the organic phase was dried with sodium sulfate and concentratedunder vacuo. The crude material was dissolved in DCM (0.2M),triethylamine (1.8 equiv.) was added, followed by Boc₂O (1.2 equiv.).The reaction was stirred for 40 min, then concentrated to dryness. Thecrude material was purified via silica gel column chromatography elutingwith hexane and ethyl acetate (1:1) to afford (±)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylateas a white foam in 66% yield. LC/MS=376.0 (M+H), LC: 3.424 min.

Synthesis of (±)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylate

To a degassed solution of (±)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate(1.0 equiv.) in MeOH and EtOAc (1:1, 0.1M) was added Pd/C (0.1 equiv.).The reaction was stirred under a hydrogen balloon overnight. Uponcompletion, the solution was filtered through a pad of Celite and thepad was washed with ethyl acetate. The filtrate was concentrated undervacuo to give (±)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylateas the desired product as a yellow foam in 93% yield. LC/MS=348.1 (M+H),Rt=055 min.

Synthesis oftert-butyl(2R)-1-(benzyloxy)-3-hydroxy-4-methylhex-5-en-2-ylcarbamate

To a solution of N-Boc, O-benzyl-D-Serine aldehyde (1.0 equiv) in DCM(0.1 M) at −78° C. under an Ar atmosphere was added(Z)-2-(but-2-enyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.1 equiv)and the clear solution stirred for 16 hours as it warmed to rt. Thesolution was added to EtOAc and was washed with H₂O (3×), andNaCl_((sat.)), dried over MgSO₄ and purified by silica gelchromatography (15% EtOAc/hexanes) to yieldtert-butyl(2R)-1-(benzyloxy)-3-hydroxy-4-methylhex-5-en-2-ylcarbamate(54%) as a 3:1 mixture of isomers as judged by ¹H NMR. LCMS (m/z): 236.3(MH⁺-Boc); LC R_(t)=4.37 and 4.51 min.

Synthesis of (4R)-4-(benzyloxymethyl)-5-(but-3-en-2-yl)oxazolidin-2-one

To a solution of (2R)-1-(benzyloxy)-3-hydroxy-4-methylhex-5-en-2-in THF(0.1 M) was added 60% sodium hydride in mineral oil (1.5 equiv.). Afterstirring for 3 days, the reaction was quenched by addition ofNH₄Cl_((sat.)) and solution was diluted with EtOAc and washed withNH₄Cl_((sat.)) and NaCl_((sat.)), dried over MgSO₄ and purified bysilica gel chromatography (50% EtOAc/hexanes) to yield(4R)-4-(benzyloxymethyl)-5-(but-3-en-2-yl)oxazolidin-2-one (89%) as a3:1 mixture. LCMS (m/z): 262.2 (MH⁺); LC R_(t)=3.47 min.

Synthesis of(4R)-4-(benzyloxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one

To a solution of(4R)-4-(benzyloxymethyl)-5-(but-3-en-2-yl)oxazolidin-2-one (1.0 equiv.)in 2:1 MeOH/H₂O (0.04 M) was added osmium tetroxide 4% in H₂O (0.07equiv) and sodium periodate (3.0 equiv.). After stirring for 3 hours,the white precipitate was filtered and rinsed with EtOAc. The combinedfiltrate was concentrated in vacuo and the residue was dissolved inEtOAc, washed with NaCl_((sat.)), dried over MgSO₄, filtered andconcentrated. The crude aldehyde was dissolved in EtOH (0.08 M) and uponcooling to 0° C., sodium borohydride (2.0 equiv.) was added. Afterstirring for 15 hours and coming to room temperature the reaction wasquenched by addition of H₂O. After stirring for 20 minutes, the EtOH wasremoved in vacuo, EtOAc was added and the solution was washed with 1NHCl, NaHCO_(3(sat.)) and NaCl_((sat.)), dried over MgSO₄, filtered andconcentrated yielding after purification by silica gel chromatography(4R)-4-(benzyloxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one as a3:1 mixture of isomers (60%). LCMS (m/z): 266.1 (MH⁺); LC R_(t)=2.28min.

Synthesis of(4R)-4-(hydroxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one

To a solution of(4R)-4-(benzyloxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one (1.0equiv.) in methanol, at a concentration of 0.1 M, was added 10%palladium on carbon (0.1 eq.). The resultant heterogeneous solution wasput under an atmosphere of hydrogen and was stirred for 15 hours. Atthis time the mixture was filtered through a pad of celite eluting withmethanol. The volatiles were removed in vacuo yielding(4R)-4-(hydroxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one (99%).LCMS (m/z): 176.1 (MH⁺).

Synthesis of 2-((4R)-2-oxo-4-(tosyloxymethyl)oxazolidin-5-yl)propyl4-methylbenzenesulfonate

To a solution of(4R)-4-(hydroxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one (1.0equiv.) in pyridine (0.15 M) at 0° C. was addedp-toluenesulfonylchloride (2.1 equiv.). The solution was allowed to warmto rt as it stirred for 14 hours, at which time EtOAc was added and thesolution was washed with H₂O(3×), CuSO_(4(sat.)) (2×), H₂O,Na₂CO_(3(sat.)) and NaCl_((sat.)), dried over MgSO₄, filtered,concentrated and purified by silica gel chromatography (75%EtOAc/hexanes eluant) yielding2-((4R)-2-oxo-4-(tosyloxymethyl)oxazolidin-5-yl)propyl4-methylbenzenesulfonate (68%). LCMS (m/z): 484.1 (MH⁺); LC R_(t)=4.06min.

Synthesis of(3aR,7R,7aS)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-oneand(3aR,7S,7aR)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one

A solution of 2-((4R)-2-oxo-4-(tosyloxymethyl)oxazolidin-5-yl)propyl4-methylbenzenesulfonate (1.0 equiv.), diisopropylethyl amine (3.0equiv.) and para-methoxybenzylamine (1.5 equiv.) in NMP (0.05 M) washeated at 100° C. for 14 hours. The solution was purified directly by RPHPLC. The product fractions were desalted by addition to EtOAc andNa₂CO_(3(s),) washed further with NaCl(sat.), dried over MgSO₄ andconcentrated yielding two separate isomers of(3aR,7R,7aS)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-oneand(3aR,7S,7aR)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one(27% and 8%). LCMS (m/z): 277.2 (MH⁺).

Synthesis of(3aR,7R,7aS)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one

To a solution of(3aR,7R,7aS)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one(1.0 equiv.) in methanol, at a concentration of 0.1 M, was added 20%palladium hydroxide on carbon (0.3 eq.). The resultant heterogeneoussolution was put under an atmosphere of hydrogen and was stirred for 2hours. At this time the mixture was filtered through a pad of celiteeluting with methanol. The volatiles were removed in vacuo yielding(3aR,7R,7aS)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one (99%).LCMS (m/z): 157.1 (MH⁺).

Synthesis of (3aR,7R,7aS)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate

A solution of 4-chloro-3-nitropyridine (1.3 equiv.) and(3aR,7R,7aS)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one (1.5equiv.) in CH₂Cl₂, at a concentration of 0.1 M, was stirred at rt for 48hours at which piperidine (0.4 equiv) was added to consume excess4-chloro-3-nitropyridine. After stirring for an additional 2 hours,di-tert-butyl dicarbonate (2.0 equiv.) and dimethylaminopyridine (0.1equiv.) were added. After stirring for 4 hours, the solution waspartitioned between EtOAc and NaHCO_(3 (sat.)), was washed further withNaHCO_(3 (sat.)), and NaCl_((sat.)), was dried over MgSO₄, was filteredand purified by silica gel chromatography yielding(3aR,7R,7aS)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)carboxylate(62%). LCMS (m/z): 379.0 (MH⁺).

Synthesis of (3aR,7R,7aS)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate

To a solution of (3aR,7R,7aS)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate(1.0 equiv.) in methanol, at a concentration of 0.1 M, was added 10%palladium on carbon (0.1 eq.). The resultant heterogeneous solution wasput under an atmosphere of hydrogen and was stirred for 14 hours. Atthis time the mixture was filtered through a pad of celite eluting withmethanol. The volatiles were removed in vacuo yielding(3aR,7R,7aS)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate.LCMS (m/z): 349.1 (MH⁺); LC R_(t)=2.06 min.

Synthesis of(3aR,7S,7aR)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one

To a solution of(3aR,7S,7aR)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one(1.0 equiv.) in methanol, at a concentration of 0.1 M, was added 20%palladium hydroxide on carbon (0.3 eq.). The resultant heterogeneoussolution was put under an atmosphere of hydrogen and was stirred for 2hours. At this time the mixture was filtered through a pad of celiteeluting with methanol. The volatiles were removed in vacuo yielding(3aR,7S,7aR)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one (99%).LCMS (m/z): 157.1 (MH⁺).

Synthesis of (3aR,7S,7aR)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate

A solution of 4-chloro-3-nitropyridine (1.3 equiv.) and(3aR,7S,7aR)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one (1.5equiv.) in CH₂Cl₂, at a concentration of 0.1 M, was stirred at rt for 48hours at which piperidine (0.4 equiv) was added to consume excess4-chloro-3-nitropyridine. After stirring for an additional 2 hours,di-tert-butyl dicarbonate (2.0 equiv.) and dimethylaminopyridine (0.1equiv.) were added. After stirring for 4 hours, the solution waspartitioned between EtOAc and NaHCO_(3 (sat.)), was washed further withNaHCO_(3 (sat.)), and NaCl_((sat.)), was dried over MgSO₄, was filteredand purified by silica gel chromatography (75% EtOAc/hexanes eluant)yielding (3aR,7S,7aR)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate(35%). LCMS (m/z): 379.0 (MH⁺). LC R_(t)=2.42 min.

Synthesis of (3aR,7R,7aS)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate

To a solution of (3aR,7S,7aR)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate(1.0 equiv.) in methanol, at a concentration of 0.1 M, was added 10%palladium on carbon (0.1 eq.). The resultant heterogeneous solution wasput under an atmosphere of hydrogen and was stirred for 14 hours. Atthis time the mixture was filtered through a pad of celite eluting withmethanol. The volatiles were removed in vacuo yielding(3aR,7S,7aR)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate.LCMS (m/z): 349.1 (MH⁺); LC R_(t)=2.18 min.

Method 3 Synthesis of 2,6-difluorobenzothioamide

A solution of 2,6 difluorobenzamide (1 eq) and Lawesson's reagent (0.5eq.) in toluene (0.2 M) was heated at 90° C. for 14 hours. Upon coolingthe volatiles were removed in vacuo and purified by SiO₂ chromatography(25% EtOAc/hexanes) yielding 2,6-difluorobenzothioamide as a lightyellow solid (99%). LCMS (m/z): 174.1 (MH⁺); LC R_(t)=2.19 min.

Synthesis of cyclohexanecarbothioamide

Following Method 3, cyclohexanecarboxamide and Lawesson's reagent werereacted, yielding cyclohexanecarbothioamide. LCMS (m/z): 144.1 (MH⁺); LCR_(t)=5.10 min.

Method 4 Synthesis of ethyl 2-(2,6-difluorophenyl)thiazole-4-carboxylate

A solution of 2,6-difluorobenzothioamide (1.0 eq) and ethylbromopyruvate(1.0 eq.) in ethanol (1.0 M) was heated in the microwave at 130° C. for30 minutes. Upon removal of volatiles in vacuo, ethyl acetate was addedand the solution was washed with Na₂CO_(3(sat.),) with NaCl_((sat.)),was dried over MgSO₄, filtered and concentrated yielding ethyl2-(2,6-difluorophenyl)thiazole-4-carboxylate (84%). LCMS (m/z): 270.1(MH⁺); LC R_(t)=3.79 min.

Synthesis of ethyl 2-cyclohexylthiazole-4-carboxylate

Following Method 4, cyclohexanecarbothioamide was used to yield ethyl2-cyclohexylthiazole-4-carboxylate. LCMS (m/z): 240.1 (MH⁺); LCR_(t)=3.90 min.

Method 5 Synthesis of 2-(2,6-difluorophenyl)thiazole-4-carboxylic acid

To a solution of ethyl 2-(2,6-difluorophenyl)thiazole-4-carboxylate (1.0eq.) in 2:1 THF/MeOH (0.17 M) was added 1.0 M LiOH (2.0 eq.). Afterstanding for 16 hours, 1.0 M HCl (2.0 eq.) was added and the THF/MeOHwas removed in vacuo. The resulting solid was filtered, rinsed with H₂Oand dried, yielding 2-(2,6-difluorophenyl)thiazole-4-carboxylic acid(88%) as a crusty solid. LCMS (m/z): 251.1 (MH⁺); LC R_(t)=2.68 min.

Synthesis of 2-cyclohexylthiazole-4-carboxylic acid

Following Method 5, ethyl 2-cyclohexylthiazole-4-carboxylate washydrolyzed yielding 2-cyclohexylthiazole-4-carboxylic acid. LCMS (m/z):212.1 (MH⁺); LC R_(t)=2.90 min.

Synthesis of ethyl 2-amino-2-cyanoacetate

To a solution of ethyl 2-cyano-2-(hydroxyimino)acetate (1 eq) in 70 mLof water and 56 mL of aq. sat. sodium bicarbonate was added portionwisethroughout 10 minutes Na₂S₂O₄ (2.8 eq) The reaction mixture was stirredat room temperature for 1 hour. The solution was saturated with sodiumchloride, extracted with methylene chloride (300 mL×3) and then thecombined organic layers were dried over anhydrous Na₂SO₄, filtered, andconcentrated in vacuo to give the titled compound, which was used tonext step without further (55%). LC/MS (m/z): 129.0 (MH⁺), R_(t): 0.25min.

Synthesis of ethyl 2-cyano-2-(2,6-difluorobenzamido)acetate

To a solution of ethyl 2-amino-2-cyanoacetate (1 eq) in 6 mL ofdichloromethane was added pyridine (1.5 eq) and 2,6-difluorobenzoylchloride (1 eq) at 0° C. The reaction mixture was stirred at roomtemperature for 3 hours. The mixture was diluted with ethyl acetate,washed with brine, then dried over anhydrous MgSO₄, filtered, andconcentrated in vacuo. The crude residue was purified by flashchromatography (EtOAc:hexanes=1:1) to give the titled compound (84%).LC/MS (m/z): 269.1 (MH⁺), R_(t): 0.69 min.

Synthesis of 5-amino-2-(2,6-difluorophenyl)thiazole-4-carboxylic acid

To a solution of the ethyl 2-cyano-2-(2,6-difluorobenzamido)acetate (1eq) in 10 mL of toluene was added Lawesson reagent. The mixture wasstirred at 95° C. for 2 days. Solvents were removed under reducedpressure. The crude residue was purified by flash chromatography(EtOAc:hexanes=1:1) to give the ethyl5-amino-2-(2,6-difluorophenyl)thiazole-4-carboxylate, which wasdissolved in 5 mL of methanol and 5 mL of THF. Then the mixture wasadded 1M sodium hydroxide (2 eq). The reaction mixture was stirred atroom temperature overnight. The reaction was concentrated to remove mostof solvents. The residue was extracted with ethyl acetate. The aqueouslayer was acidified to pH=4-5 by 1N HCl. The resulting mixture wasextracted by ethyl acetate. The organic layer was separated, washed withbrine, then dried over anhydrous MgSO₄, filtered, and concentrated invacuo. to give the pure titled compound (34%). LC/MS (m/z): 257.1 (MH⁺),R_(t): 0.61 min.

Method 6 Synthesis of(S)-N-(4-(3-aminopiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide

A homogeneous solution of 1 eq each of (S)-tert-butyl1-(3-aminopyridin-4-yl)piperidin-3-ylcarbamate,2-(2,6-difluorophenyl)thiazole-4-carboxylic acid, HOAT and EDC in NMP,at a concentration of 0.38 M, was left standing for 48 hours at whichtime the mixture was directly purified by HPLC. Upon lyophilization, theTFA salt of (S)-tert-butyl1-(3-(2-(2,6-difluorophenyl)thiazole-4-carboxamido)pyridin-4-yl)piperidin-3-ylcarbamatewas obtained. Alternatively, the HPLC fractions could be added to EtOAcand solid Na₂CO₃, separated and washed with NaCl_((sat.).) Upon dryingover MgSO₄, filtering and removing the volatiles in vacuo,(S)-tert-butyl1-(3-(2-(2,6-difluorophenyl)thiazole-4-carboxamido)pyridin-4-yl)piperidin-3-ylcarbamatewas obtained.

The Boc group was removed by treating with 25% TFA/CH₂Cl₂ for 2 hours orwith excess 4M HCl in dioxane for 12 hours. Upon removal of thevolatiles in vacuo, the material was purified by RP HPLC yielding afterlyophilization(S)-N-(4-(3-aminopiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamideas the TFA salt. Alternatively, the HPLC fractions could be added toEtOAc and solid Na₂CO₃, separated and washed with NaCl_((sat.).) Upondrying over MgSO₄, filtering and removing the volatiles in vacuo thefree base was obtained. Upon dissolving in MeCN/H₂O, adding 1 eq. of 1 NHCl and lyophilizing, the HCl salt of(S)-N-(4-(3-aminopiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamidewas obtained (43%). LCMS (m/z): 416.1 (MH⁺); LC R_(t)=1.95 min.

If benzoyl protected hydroxyls were present they could be deprotectedprior to Boc removal by treating with 0.2 M sodium hydroxide (3 eq) inMeOH at room temperature for 3 hours, upon which time the solution wasdiluted with ethyl acetate, washed with brine, dried over anhydrousMgSO₄, filtered, and concentrated in vacuo to yield the Boc protectedalcohol.

If an N-Boc1,2 amino alcohol cyclic carbamate was present, prior to Bocdeprotection the cyclic carbamate could be cleaved by treating withCs₂CO₂ (0.5 eq) in methanol at a concentration of 0.1 M for three hours.After removal of volatiles in vacuo, the Boc amino group was deprotectedas described above.

If TBDMS ethers were present they were deprotected prior to Boc removalby treating with 6N HCl, THF, methanol (1:2:1) at room temperature for 2h. After removal of volatiles in vacuo, the Boc amino group wasdeprotected as described above.

If a diethoxyphosphorylamino group was present, the amine wasdeprotected by heating in a 1:1 solution of dioxane/2N HCl_((aq.)) at70° C. overnight. Upon removal of the volatiles in vacuo, the materialwas purified by RP HPLC.

The following compounds were prepared using Method 6:

LC/MS LC/MS (M + H (Rf min Ex. on on No. Structure Compound Name UPCL)UPLC) 1

(S)-N-(4-(3-aminopiperidin- 1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 416.1 1.95 2

N-(4-((3R,4R)-3-amino-4- hydroxypiperidin-1-yl) pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 432.1 1.84 3

N-(4-((3R,4R)-3-amino-4- fluoropiperidin-1-yl) pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 434.1 2.23 4

N-(4-((3S,4S)-3-amino-4- fluoropiperidin-1-yl) pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 434.1 2.22 5

N-(4-((3S,5R)-3-amino-5- hydroxypiperidin-1-yl) pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 432 1.65 6

N-(4-((3R,4S)-3-amino-4- hydroxypiperidin-1-yl) pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 432.1 1.89 7

N-(4-(3-amino-4- methoxypiperidin-1-yl) pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 445.9 1.96 8

N-(4-((3S,5R)-3-amino-5- methoxypiperidin-1-yl) pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 446.1 1.83 9

N-(4-((3S,5S)-3-amino-5- hydroxypiperidin-1-yl) pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 432.0 1.75 10

(3S,5S)-5-amino-1-(3-(2- (2,6-difluorophenyl) thiazole-4-carboxamido)pyridin-4-yl) piperidin-3-yl benzoate 536.1 2.44 11

N-(4-((3S,5R)-3-amino-5- fluoropiperidin-1-yl) pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 433.9 1.80 12

(S)-N-(4-(3-aminopiperidin- 1-yl) pyridin-3-yl)-2- cyclohexylthiazole-4-carboxamide 386.1 2.12 13

N-(4-(3-amino-5- (hydroxymethyl)piperidin- 1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 446.0 1.78 14

N-(4-(3-amino-5- methylpiperidin-1- yl)pyridin-3-yl)- 2-(2,6-difluorophenyl)thiazole- 4-carboxamide 430.0 2.14 15

N-(4-(3-amino-5- (trifluoromethyl) piperidin-1-yl)pyridin-3-yl)- 2-(2,6-difluorophenyl)thiazole- 4-carboxamide 484.1 2.27 16

N-(4-(3-amino-5- (fluoromethyl)piperidin- 1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 448.0 2.05 17

N-(4-((3S,5S)-3-amino-5- fluoropiperidin-1- yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 434.0 0.49 18

N-(4-(3-amino-4- chloropiperidin- 1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 449.8 0.55 19

N-(4-(3-amino-4- methylpiperidin- 1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 430.1 0.57 20

N-(4-(3-amino-5- methylpiperidin- 1-yl)pyridin-3-yl)-2-cyclohexylthiazole-4- carboxamide 400.1 0.64 21

N-(4-(3-amino-5- (trifluoromethyl)piperidin- 1-yl)pyridin-3-yl)-2-cyclohexylthiazole-4- carboxamide 454.1 0.72 22

N-(4-((3S,5S)-3-amino-5- methoxypiperidin-1- yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 446.1 0.54 23

N-(4-((3S,5R)-3-amino-5- ethoxypiperidin-1- yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 460.0 0.54 24

(S)-N-(4-(5-amino-3,3- difluoropiperidin-1- yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 452.0 0.56 25

N-(4-((3S,5R)-3-amino-5- ethylpiperidin-1-yl)pyridin- 3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 444.1 0.57 26

N-(4-((3R,5R)-3-amino-5- (trifluoromethyl)piperidin- 1-yl)pyridin-3-yl)-2-(2,6- difluorophenyl)thiazole- 4-carboxamide 484.1 0.57 27

N-(4-((3S,5R)-3-amino-5- (trifluoromethyl)piperidin- 1-yl)pyridin-3-yl)-2-(2,6- difluorophenyl)thiazole- 4-carboxamide 484.0 0.68 28

N-(4-((3S,5R)-3-amino-5- methylpiperidin-1- yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole- 4-carboxamide 430.2 0.60 29

(S)-5-amino-N-(4-(3- aminopiperidin-1- yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 431.0 0.51 30

5-amino-2-(2,6- difluorophenyl)-N-(4-(4- fluoropiperidin-1-yl)pyridin-3-yl)thiazole-4- carboxamide 434.0 0.75 31

2-(2,6-difluorophenyl)-N- (4-((1R,3S,5S)-3-hydroxy- 5-methylcyclohexyl)pyridin- 3-yl)thiazole-4-carboxamide 430.1 0.7 32

5-amino-2-(2,6- difluorophenyl)-N-(4- ((1R,3S,5S)-3-hydroxy-5-methylcyclohexyl)pyridin- 3-yl)thiazole-4-carboxamide 445.1 0.7 33

5-amino-N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3- yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 430.0 0.54 34

N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3- yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 415.0 0.54 35

N-(4-((3R,4S,5R)-3-amino- 4-hydroxy-5- methylpiperidin-1-yl)pyridin-3-yl)-2-(2,6- difluorophenyl)thiazole-4- carboxamide 446.20.51 36

N-(4-((3R,4R,5S)-3-amino- 4-hydroxy-5- methylpiperidin-1-yl)pyridin-3-yl)-2-(2,6- difluorophenyl)thiazole-4- carboxamide 446.00.55 37

N-(4-((1R,3R,4S)-3-amino- 4- hydroxycyclohexyl)pyridin- 3-yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 431.0 0.50 38

5-amino-N-(4-(3-amino-4- hydroxycyclohexyl)pyridin- 3-yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 446.1 0.51 39

N-(4-((1R,3R,4R)-3-amino- 4- hydroxycyclohexyl)pyridin- 3-yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 431.0 0.54 40

N-(4-((1R,3S,5S)-3-amino- 5- methylcyclohexyl)pyridin-3-yl)-2-cyclohexylthiazole- 4-carboxamide 399.3 0.63 41

N-(4-((1R,3R,4R,5S)-3- amino-4-hydroxy-5- methylcyclohexyl)pyridin-3-yl)-2-(2,6- difluorophenyl)thiazole-4- carboxamide (and enantiomer)445.1 0.52 42

N-(4-((1R,3R,4R,5S)-3- amino-4-hydroxy-5- methylcyclohexyl)pyridin-3-yl)-2-cyclohexylthiazole- 4-carboxamide 415.1 0.60 43

N-(4-((1R,3R,4S,5S)-3- amino-4-hydroxy-5- methylcyclohexyl)pyridin-3-yl)-2-(2,6- difluorophenyl)thiazole-4- carboxamide (and enantiomer)445.2 0.54 44

N-(4-((1S,3R,5R)-3-amino- 5- methylcyclohexyl)pyridin- 3-yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 429.2 0.68 45

N-(4-((1R,3S,5S)-3-amino- 5- methylcyclohexyl)pyridin- 3-yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 429.2 0.68 46

N-(4-((1R,5R)-5-amino-3,3- dimethylcyclohexyl)pyridin- 3-yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 443.2 0.60 47

N-(4-((1R,5R)-5-amino-3,3- dimethylcyclohexyl)pyridin- 3-yl)-2-(2,6-difluorophenyl)thiazole-4- carboxamide 443.2 0.60

Synthesis of (S)-tert-butyl1-(3-(2-bromothiazole-4-carboxamido)pyridin-4-yl)piperidin-3-ylcarbamate

A solution containing 1 eq each of (S)-tert-butyl1-(3-aminopyridin-4-yl)piperidin-3-ylcarbamate,2-bromothiazole-4-carboxylic acid, HOAT and EDC in DMF, at aconcentration of 0.5 M, was stirred for 60 hours. The solution wasdiluted with EtOAc and was washed with H₂O (4×), NaCl_((sat.)), wasdried over MgSO₄, was filtered and the volatiles were removed in vacuoyielding (S)-tert-butyl1-(3-(2-bromothiazole-4-carboxamido)pyridin-4-yl)piperidin-3-ylcarbamate,LCMS (m/z): 416.1 (MH⁺); LC R_(t)=1.95 min.

Method 7 Synthesis of(S)-N-(4-(3-aminopiperidin-1-yl)pyridin-3-yl)-2-(2-fluorophenyl)thiazole-4-carboxamide

A solution of (S)-tert-butyl1-(3-(2-bromothiazole-4-carboxamido)pyridin-4-yl)piperidin-3-ylcarbamate(1.0 eq), 2-fluorophenyl boronic acid (3.0 eq.), Pd(dppf)Cl₂—CH₂Cl₂(0.15 eq.) in 3:1 DME/2M Na₂CO₃ (concentration=0.1 M) was heated at 120°C. with microwave irradiation for 1200 seconds. Upon cooling the organiclayer was separated, concentrated and the N-Boc Suzuki product wasdirectly purified by reverse phase HPLC. The product fraction waslyophilized and the resulting solid was treated with 25% TFA/DCM (at aresulting concentration of 0.05 M). After sitting for 2 hours, thevolatiles were removed in vacuo and the residue was purified by reversephase HPLC. After lyophilization,(S)-N-(4-(3-aminopiperidin-1-yl)pyridin-3-yl)-2-(2-fluorophenyl)thiazole-4-carboxamidewas obtained (35%) as the TFA salt. LCMS (m/z): 398.1 (MH⁺); LC Rt=2.06min.

The following compounds were prepared using Method 7:

LC/MS LC/MS (M + H (Rf min Ex. on on No. Structure Compound Name UPCL)UPLC) 48

(S)-N-(4-(3- aminopiperidin-1- yl)pyridin-3-yl)- 2-phenylthiazole-4-carboxamide 380.1 1.94 49

(S)-N-(4-(3- aminopiperidin-1- yl)pyridin-3-yl)-2-(2- fluorophenyl)thiazole-4- carboxamide 398.1 2.06 50

(S)-N-(4-(3- aminopiperidin-1- yl)pyridin-3-yl)- 2-(2-fluoro-5-(isopropylcarbamoyl) phenyl)thiazole- 4-carboxamide 483.2 2.04 51

N-(4-((3S,4S)-3- amino-4-hydroxy- piperidin-1-yl) pyridin-3-yl)-2-(2,6-difluoro phenyl)thiazole-4- carboxamide 432.1 1.85

Synthesis oftert-butyl(3R,4R)-1-(3-(2-bromothiazole-4-carboxamido)pyridin-4-yl)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate

A solution containing 1 eq each oftert-butyl(3R,4R)-1-(3-aminopyridin-4-yl)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate,2-bromothiazole-4-carboxylic acid, HOAT and EDC in DMF, at aconcentration of 0.5 M, was stirred for 60 hours. The solution wasdiluted with EtOAc and was washed with H₂O (4×), NaCl_((sat.)), wasdried over MgSO₄, was filtered and the volatiles were removed in vacuoyieldingtert-butyl(3R,4R)-1-(3-(2-bromothiazole-4-carboxamido)pyridin-4-yl)-4-(tert-butyldimethylsilyloxy)piperidin-3-ylcarbamate,LCMS (m/z): 612.2/614.2 (MH⁺); LC R_(t)=4.26 min.

Synthesis oftert-butyl(1S,3R,5S)-3-(3-(2-bromothiazole-4-carboxamido)pyridin-4-yl)-5-methylcyclohexylcarbamate

A solution containing 1 eq each oftert-butyl(1S,3R,5S)-3-(3-aminopyridin-4-yl)-5-methylcyclohexylcarbamate,2-bromothiazole-4-carboxylic acid, HOAT and EDC in DMF, at aconcentration of 0.3 M, was stirred for 17 hours. The solution wasdiluted with EtOAc and was washed with H₂O (4×), NaCl_((sat.)), wasdried over MgSO₄, was filtered and the volatiles were removed in vacuoyielding tert-butyl(1S,3R,5S)-3-(3-(2-bromothiazole-4-carboxamido)pyridin-4-yl)-5-methylcyclohexylcarbamate,LCMS (m/z): 495.1/497.1 (MH⁺); LC R_(t)=3.17 min.

Method 8 Synthesis ofN-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluoro-3-methoxyphenyl)thiazole-4-carboxamide

A solution oftert-butyl(1S,3R,5S)-3-(3-(2-bromothiazole-4-carboxamido)pyridin-4-yl)-5-methylcyclohexylcarbamate(1.0 equiv.), 2,6-difluoro-3-methoxyphenylboronic acid (4.0 equiv.),DIEA (4.0 equiv.) and Pd(PPh₃)₄ (0.2 equiv) in 1:1 toluene/ethanol at aconcentration of 0.03 M was heated in a microwave at 120° C. for 20minutes. The solution was resubmitted to heating in the microwave at130° C. for 2×30 min. The solvents were removed in vacuo and the residuewas purified by HPLC. The product fractions were lyophilized directly toyield the protected amide product as the TFA salt. The Boc group wasdeprotected by treating with 25% TFA/CH₂Cl₂ for two hours. Upon removalof volatiles in vacuo, the product was purified by HPLC to yieldN-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluoro-3-methoxyphenyl)thiazole4-carboxamide.LCMS (m/z): 459.2 (MH⁺); LC R_(t)=2.32 min.

When applying the above method for Suzuki it is at times necessary toresubmit to heating in the microwave and additionally add more of theboronic acid (4.0 equiv.), DIEA (4.0 equiv.) and Pd(PPh₃)₄ (0.2 equiv)with each resubmission.

Alternatively, Suzuki reactions with 2,6 difluorosubstituted boronicacids and 2-bromo-4-carboxamido thiazoles can be carried out by heatingthe bromide (1.0 equiv), boronic acid (5 equiv.), KF (5.5 equiv.),tri-tert-butylphosphine (0.4 equiv) and Pd₂(dba)₃ (0.2 equiv.) in 10:1THF/H₂O at a concentration of 0.03 M in the microwave at 100° C. for 30minutes. With this method, is at times necessary to resubmit to heatingin the microwave at 100° C. and additionally add more of the boronicacid (5.0 equiv.), KF (5.5 equiv.), tri-tert-butylphosphine (0.4 equiv)and Pd₂(dba)₃ (0.2 equiv.) with each resubmission.

The following compounds were prepared using Method 8:

LC/MS LC/MS (M + H (Rf min Ex. on on No. Structure Compound Name UPCL)UPLC) 52

2-(2,6-Difluoro-3- methoxy-phenyl)- thiazole-4-carboxylic acid[4-(3-amino-5- methyl-cyclohexyl)- pyridin-3-yl]-amide 459.2 0.58 53

2-[2-Fluoro-1-(1- fluoro-vinyl)-3- isopropoxy- propenyl]-thiazole-4-carboxy acid [4- (3-amino-5-methyl- cyclohexyl)-pyridin- 3-yl]-amide487.3 0.67 54

2-(3-Ethoxy-2,6- difluoro-phenyl)- thiazole-4-carboxylic acid[4-(3-amino-5- methyl-cyclohexyl)- pyridin-3-yl]-amide 473.3 0.63 55

2-(2,3,6-Trifluoro- phenyl)-thiazole-4- carboxylic acid [4-(3-amino-5-methyl- cyclohexyl)-pyridin- 3-yl]-amide 447.2 0.59 56

2-(3-Chloro-2,6- difluoro-phenyl)- thiazole-4-carboxylic acid[4-(3-amino-5- methyl-cyclohexyl)- pyridin-3-yl]-amide 463.1 0.62 57

2-(2,6-Difluoro-4- hydroxy-phenyl)- thiazole-4-carboxylic acid[4-(3-amino-5- methyl-cyclohexyl)- pyridin-3-yl]-amide 445.2 0.54

Example 58 Synthesis ofN-(4-((±)-3-amino-4-fluoro-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide

To a solution of(±)-2-(tert-butoxycarbonylamino)-4-(3-(2-(2,6-difluorophenyl)thiazole-4-carboxamido)pyridin-4-yl)-6-methylcyclohexylacetate in MeOH (0.03 M) was added potassium carbonate (3.0 equiv.) andthe reaction was stirred at room temperature for 3 h. Upon completion,the reaction was partitioned between water and ethyl acetate. Theaqueous phase was further extracted 3 more times with ethyl acetate. Theorganics were combined, dried with brine and sodium sulfate, filtered,and concentrated. Isolatedtert-butyl(±)-5-(3-(2-(2,6-difluorophenyl)thiazole-4-carboxamido)pyridin-4-yl)-2-hydroxy-3-methylcyclohexylcarbamatewas obtained as an off-white solid in 87% yield. LC/MS=545.2 (M+H),Rt=0.75 min. To a solution oftert-butyl(±)-5-(3-(2-(2,6-difluorophenyl)thiazole-4-carboxamido)pyridin-4-yl)-2-hydroxy-3-methylcyclohexylcarbamate(1.0 equiv.) in DCM (0.07M) at 0° C. was added DAST (1.0 equiv.) under anitrogen atmosphere. The reaction was stirred for 1 h, upon which time,2 more equivalents of DAST were added. After 30 min, the consumption ofthe starting material was complete. The reaction was quenched by theaddition of water, the organic phase was dried with sodium sulfate, andconcentrated. The crude material was purified via silica gel columnchromatography eluting with ethyl acetate and hexanes (1:1) to affordtert-butyl(±)-5-(3-(2-(2,6-difluorophenyl)thiazole-4-carboxamido)pyridin-4-yl)-2-fluoro-3-methylcyclohexylcarbamateas a clear oil in 60% yield. LC/MS=547.3 (M+H), Rt=0.92 min. The Bocgroup was removed by treating with 25% TFA in dichloromethane for 2hours. Upon concentration under vacuo, the crude was purified viasemi-prep HPLC. The pure fractions were lyophilized to yieldN-(4-((±)-3-amino-4-fluoro-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamideas the desired product as the TFA salt in 74% yield. LC/MS=447.0 (M+H),Rt=0.56 min.

The following compounds are prepared using the techniques and proceduresdescribed herein.

Example Structure Name 59

2-(2,6-Difluoro-3-methyl-phenyl)- thiazole-4-carboxylic acid (3-amino-4-hydroxy-5-methyl-3,4,5,6- tetrahydro-2H-[1,4′]bipyridinyl-3′-yl)-amide 60

2-(2-Fluoro-5-methyl-phenyl)- thiazole-4-carboxylic acid (3-amino-4-hydroxy-5-methyl-3,4,5,6- tetrahydro-2H-[1,4′]bipyridinyl-3′-yl)-amide 61

2-(2,3,6-Trifluoro-phenyl)-thiazole- 4-carboxylic acid (3-amino-4-hydroxy-5-methyl-3,4,5,6- tetrahydro-2H-[1,4′]bipyridinyl-3′- yl)-amide62

2-(2-Fluoro-phenyl)-thiazole-4- carboxylic acid (3-amino-4-hydroxy-5-methyl-3,4,5,6-tetrahydro-2H- [1,4′]bipyridinyl-3′-yl)-amide 63

2-(2-Chloro-6-fluoro-phenyl)- thiazole-4-carboxylic acid (3-amino-4-hydroxy-5-methyl-3,4,5,6- tetrahydro-2H-[1,4′]bipyridinyl-3′-yl)-amide 64

2-(2,6-Difluoro-3-methoxy-phenyl)- thiazole-4-carboxylic acid (3-amino-4-hydroxy-5-methyl-3,4,5,6- tetrahydro-2H-[1,4′]bipyridinyl-3′-yl)-amide 65

2-Phenyl-thiazole-4-carboxylic acid (3-amino-4-hydroxy-5-methyl-3,4,5,6-tetrahydro-2H- [1,4]bipyridinyl-3′-yl)-amide 66

2-(2-Chloro-phenyl)-thiazole-4- carboxylic acid (3-amino-4-hydroxy-5-methyl-3,4,5,6-tetrahydro-2H- [1,4′]bipyridinyl-3′-yl)-amide 67

2-(2,6-Difluoro-3-hydroxy-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-5-methyl-cyclohexyl)-pyridin- 3-yl]-amide 68

2-(2-Fluoro-5-hydroxy-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-5-methyl-cyclohexyl)-pyridin- 3-yl]-amide 69

2-(2-Fluoro-4-hydroxy-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-5-methyl-cyclohexyl)-pyridin- 3-yl]-amide 70

2-(2,6-Difluoro-4-hydroxy-phenyl)- thiazole-4-carboxylic acid [4-(5-amino-3,3-dimethyl-cyclohexyl)- pyridin-3-yl]-amide 71

2-(2,6-Difluoro-3-hydroxy-phenyl)- thiazole-4-carboxylic acid [4-(5-amino-3,3-dimethyl-cyclohexyl)- pyridin-3-yl]-amide 72

2-(2-Fluoro-5-hydroxy-phenyl)- thiazole-4-carboxylic acid [4-(5-amino-3,3-dimethyl-cyclohexyl)- pyridin-3-yl]-amide 73

2-(2-Fluoro-4-hydroxy-phenyl)- thiazole-4-carboxylic acid [4-(5-amino-3,3-dimethyl-cyclohexyl)- pyridin-3-yl]-amide 74

2-(3-Chloro-2,6-difluoro-phenyl)- thiazole-4-carboxylic acid [4-(5-amino-3,3-dimethyl-cyclohexyl)- pyridin-3-yl]-amide 75

2-(5-Chloro-2-fluoro-phenyl)- thiazole-4-carboxylic acid [4-(5-amino-3,3-dimethyl-cyclohexyl)- pyridin-3-yl]-amide 76

2-(2-Fluoro-5-methyl-phenyl)- thiazole-4-carboxylic acid [4-(5-amino-3,3-dimethyl-cyclohexyl)- pyridin-3-yl]-amide 77

2-(2,6-Difluoro-3-methyl-phenyl)- thiazole-4-carboxylic acid [4-(5-amino-3,3-dimethyl-cyclohexyl)- pyridin-3-yl]-amide 78

2-(2-Fluoro-5-methyl-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-3,3-dimethyl-cyclohexyl)- pyridin-3-yl]-amide 79

2-(2,6-Difluoro-3-methyl-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-5-methyl-cyclohexyl)- pyridin-3-yl]-amide 80

2-(2,6-Difluoro-4-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-methyl-3,4,5,6-tetrahydro-2H- [1,4′]bipyridinyl-3′-yl)-amide 81

2-(2,6-Difluoro-3-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-methyl-3,4,5,6-tetrahydro-2H- [1,4′]bipyridinyl-3′-yl)-amide 82

2-(2-Fluoro-5-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-methyl-3,4,5,6-tetrahydro-2H- [1,4′]bipyridinyl-3′-yl)-amide 83

2-(2-Fluoro-4-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-methyl-3,4,5,6-tetrahydro-2H- [1,4′]bipyridinyl-3′-yl)-amide 84

2-(2,6-Difluoro-4-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-ethyl-3,4,5,6-tetrahydro-2H- [1,4′]bipyridinyl-3′-yl)-amide 85

2-(2,6-Difluoro-3-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-ethyl-3,4,5,6-tetrahydro-2H- [1,4′]bipyridinyl-3′-yl)-amide 86

2-(2-Fluoro-5-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-ethyl-3,4,5,6-tetrahydro-2H- [1,4′]bipyridinyl-3′-yl)-amide 87

2-(2-Fluoro-4-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-ethyl-3,4,5,6-tetrahydro-2H- [1,4′]bipyridinyl-3′-yl)-amide 88

2-(2,6-Difluoro-4-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-trifluoromethyl-3,4,5,6-tetrahydro- 2H-[1,4′]bipyridinyl-3′-yl)-amide89

2-(2,6-Difluoro-3-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-trifluoromethyl-3,4,5,6-tetrahydro- 2H-[1,4′]bipyridinyl-3′-yl)-amide90

2-(2-Fluoro-5-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-trifluoromethyl-3,4,5,6-tetrahydro- 2H-[1,4′]bipyridinyl-3′-yl)-amide91

2-(2-Fluoro-4-hydroxy-phenyl)- thiazole-4-carboxylic acid (5-amino-3-trifluoromethyl-3,4,5,6-tetrahydro- 2H-[1,4′]bipyridinyl-3′-yl)-amide92

2-(2,6-Difluoro-4-hydroxy-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-cyclohexyl)-pyridin-3-yl]- amide 93

2-(2,6-Difluoro-3-hydroxy-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-cyclohexyl)-pyridin-3-yl]- amide 94

2-(2-Fluoro-5-hydroxy-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-cyclohexyl)-pyridin-3-yl]- amide 95

2-(2-Fluoro-4-hydroxy-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-cyclohexyl)-pyridin-3-yl]- amide 96

2-(3-Chloro-2,6-difluoro-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-cyclohexyl)-pyridin-3-yl]- amide 97

2-(5-Chloro-2-fluoro-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-cyclohexyl)-pyridin-3-yl]- amide 98

2-(2-Fluoro-5-methyl-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-cyclohexyl)-pyridin-3-yl]- amide 99

2-(2,6-Difluoro-3-methyl-phenyl)- thiazole-4-carboxylic acid [4-(3-amino-cyclohexyl)-pyridin-3-yl]- amide

Pim1 ATP Depletion Assay

The activity of PIM1 is measured using a luciferase-luciferin based ATPdetection reagent to quantify ATP depletion resulting fromkinase-catalyzed phosphoryl transfer to a peptide substrate. Compoundsto be tested are dissolved in 100% DMSO and directly distributed intowhite 384-well plates at 0.5 μl per well. To start the reaction, 10 μlof 5 nM Pim1 kinase and 80 μM BAD peptide (RSRHSSYPAGT-OH) in assaybuffer (50 mM HEPES pH 7.5, 5 mM MgCl₂, and 1 mM DTT, 0.05% BSA) isadded into each well. After 15 minutes, 10 μl of 40 μM ATP in assaybuffer is added. Final assay concentrations are 2.5 nM PIM1, 20 μM ATP,40 μM BAD peptide and 2.5% DMSO. The reaction is performed untilapproximately 50% of the ATP is depleted, then stopped with the additionof 20 μl KinaseGlo Plus (Promega Corporation) solution. The stoppedreaction is incubated for 10 minutes and the remaining ATP detected vialuminescence on the Victor2 (Perkin Elmer). Compounds of the foregoingexamples were tested by the Pim1 ATP depletion assay and found toexhibit an IC₅₀ values as shown in the table below. IC₅₀, the halfmaximal inhibitory concentration, represents the concentration of a testcompound that is required for 50% inhibition of its target in vitro.

Pim2 ATP Depletion Assay

The activity of PIM2 is measured using a luciferase-luciferin based ATPdetection reagent to quantify ATP depletion resulting fromkinase-catalyzed phosphoryl transfer to a peptide substrate. Compoundsto be tested are dissolved in 100% DMSO and directly distributed intowhite 384-well plates at 0.5 μl per well. To start the reaction, 10 μlof 10 nM Pim2 kinase and 20 μM BAD peptide (RSRHSSYPAGT-OH) in assaybuffer (50 mM HEPES pH 7.5, 5 mM MgCl₂, and 1 mM DTT, 0.05% BSA) isadded into each well. After 15 minutes, 10 μl of 8 μM ATP in assaybuffer is added. Final assay concentrations are 5 nM PIM2, 4 μM ATP, 10μM BAD peptide and 2.5% DMSO. The reaction is performed untilapproximately 50% of the ATP is depleted, then stopped with the additionof 20 μl KinaseGlo Plus (Promega Corporation) solution. The stoppedreaction is incubated for 10 minutes and the remaining ATP detected vialuminescence on the Victor2 (Perkin Elmer). Compounds of the foregoingexamples were tested by the Pim2 ATP depletion assay and found toexhibit an IC₅₀ values as shown in the table below.

Pim3 ATP Depletion Assay

The activity of PIM3 is measured using a luciferase-luciferin based ATPdetection reagent to quantify ATP depletion resulting fromkinase-catalyzed phosphoryl transfer to a peptide substrate. Compoundsto be tested are dissolved in 100% DMSO and directly distributed intowhite 384-well plates at 0.5 μl per well. To start the reaction, 10 μlof 10 nM Pim3 kinase and 200 μM BAD peptide (RSRHSSYPAGT-OH) in assaybuffer (50 mM HEPES pH 7.5, 5 mM MgCl₂, and 1 mM DTT, 0.05% BSA) isadded into each well. After 15 minutes, 10 μl of 80 μM ATP in assaybuffer is added. Final assay concentrations are 5 nM PIM1, 40 μM ATP,100 μM BAD peptide and 2.5% DMSO. The reaction is performed untilapproximately 50% of the ATP is depleted, then stopped by the additionof 20 μl KinaseGlo Plus (Promega Corporation) solution. The stoppedreaction is incubated for 10 minutes and the remaining ATP detected vialuminescence on the Victor2 (Perkin Elmer). Compounds of the foregoingexamples were tested by the Pim3 ATP depletion assay and found toexhibit an IC₅₀ values as shown in the table below.

Cell Proliferation Assay

KMS11 (human myeloma cell line), were cultured in IMDM supplemented with10% FBS, sodium pyruvate and antibiotics. Cells were plated in the samemedium at a density of 2000 cells per well into 96 well tissue cultureplates, with outside wells vacant, on the day of assay. MM1.s (humanmyeloma cell line), were cultured in RPMI1640 supplemented with 10% FBS,sodium pyruvate and antibiotics. Cells were plated in the same medium ata density of 5000 cells per well into 96 well tissue culture plates,with outside wells vacant, on the day of assay.

Test compounds supplied in DMSO were diluted into DMSO at 500 times thedesired final concentrations before dilution into culture media to 2times final concentrations. Equal volumes of 2× compounds were added tothe cells in 96 well plates and incubated at 37° C. for 3 days.

After 3 days plates were equilibrated to room temperature and equalvolume of Cell Titer-Glow Reagent (Promega) was added to the culturewells. The plates were agitated briefly and luminescent signal wasmeasured with luminometer. The percent inhibition of the signal seen incells treated with DMSO alone vs. cells treated with control compoundwas calculated and used to determine EC₅₀ values (i.e., theconcentration of a test compound that is required to obtain 50% of themaximum effect in the cells) for tested compounds, as shown in Table 6below.

IC50 and EC50 Activity of Compounds of the Invention

Using the procedures for Pim1 ATP depletion assay (PIM-1), Pim2 ATPdepletion assay (PIM-2), and Pim3 ATP depletion assay (PIM-3), describedabove, the IC₅₀ concentration of compounds of the previous examples weredetermined as shown in the following table.

Pim1 Pim2 Pim3 Ex. IC₅₀ IC₅₀ IC₅₀ No. Structure (μM (μM) (μM) 1

0.001 0.008 0.002 2

0.002 0.011 0.004 3

0.002 0.023 0.005 5

0.015 0.077 0.013 6

0.001 0.003 0.001 7

0.005 0.243 0.018 8

0.021 0.046 0.035 9

0.069 1.502 0.091 10

2.220 0.479 0.322 11

0.004 0.055 0.008 12

0.057 0.208 0.045 13

0.014 0.058 0.024 14

0.002 0.004 0.005 15

0.001 0.003 0.004 16

0.001 0.003 0.004 17

0.004 0.070 0.008 18

0.005 0.054 0.012 19

0.001 0.008 0.005 20

0.012 0.018 0.016 21

0.011 0.021 0.020 22

0.129 0.378 0.060 23

0.056 0.085 0.086 24

0.002 0.038 0.004 25

0.001 0.003 0.003 26

0.006 0.072 0.024 27

0.001 0.003 0.003 28

0.001 0.002 0.002 29

0.001 0.002 0.002 30

0.001 0.055 0.003 31

0.002 0.090 0.008 32

0.001 0.005 0.004 33

0.001 0.003 0.003 34

0.002 0.029 0.004 35

0.009 0.125 0.009 36

0.001 0.003 0.003 37

0.006 0.098 0.011 38

0.001 0.004 0.003 39

0.011 0.095 0.012 40

0.041 0.097 0.109 41

0.004 0.027 0.008 42

0.146 0.250 0.215 43

0.005 0.046 0.015 45

0.001 0.007 0.003 46

0.001 0.016 0.003 48

0.0020 0.0177 0.0023 49

0.0019 0.0099 0.0042 50

0.0032 0.0838 0.0083 52

0.001 0.004 0.002 53

0.001 0.020 0.003 54

0.001 0.009 0.003 55

0.003 0.015 0.005 56

0.001 0.012 0.003 57

0.001 0.004 0.002 58

0.004 0.047 0.018

Using the procedures for Cell Proliferation Assay described above, theEC₅₀ concentration of compounds of the previous examples were determinedin KMS11 cells as shown in the following table.

KMS11 Ex. EC₅₀ No. Structure (μM) 1

1.17 2

1.57 6

0.41 8

4.70 11

6.10 14

0.83 15

0.31 16

2.38 19

0.93 23

6.54 27

0.46 28

0.40 29

0.04 33

0.43 34

5.02 36

0.56 38

1.89 41

>10 42

43

45

0.92

1. A compound of Formula I, or a stereoisomer, tautomer, orpharmaceutically acceptable salt thereof,

wherein, R₁ is selected from

X represents CH, or N; R_(2a) is selected from amino, methyl, CH₂F, CF₃,C₂H₅, and H; R_(2b) is selected from H, and methyl; R₃ is selected fromH, OH, OCH₃, CH₃, F, and Cl; R_(4a) is selected from amino, methyl, OH,OCH₃, OC₂H₅, F, CF₃, H, and ethyl; R_(4b) is selected from methyl, H,and F; R₂₁ represents H or F; R₂₂ represents H, Cl, or F; R₂₃ representsF, OC₂H₅, OCH₃, Cl, H, methyl, OH, or OCH(CH₃)₂; and R₂₄ represents H orOH.
 2. A compound of Formula II, or a stereoisomer, tautomer, orpharmaceutically acceptable salt thereof,

wherein: R₁ is selected from —NH—CO-alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl; Xrepresents CH or N; R_(2a) is selected from —H, —OH, alkyl, alkoxy,haloalkyl, aminoalkyl, hydroxyalkyl, halo, amino and bezoate; R_(2b) isselected from —H and alkyl; R₃ is selected from H, OH, alkyl, alkoxy andhalo; R_(4a) is selected from —OH, alkyl, alkoxy, haloalkyl, aminoalkyl,hydroxyalkyl, halo and amino; and R_(4b) is selected from H, alkyl andhalo.
 3. A compound of claim 2 wherein R₁ is selected from substitutedor unsubstituted phenyl, substituted or unsubstituted cyclohexyl, andsubstituted or unsubstituted piperidinyl.
 4. A compound of claim 3wherein R₁ is selected from

wherein: R₂₁ is H or halo; R₂₂ is H or halo; R₂₃ is selected from H,halo, alkyl and alkoxy; and R₂₄ is H or OH;
 5. A compound of claim 4wherein R₂₁ and R₂₂ are independently selected from H or F.
 6. Acompound of claim 4 wherein R₂₃ is selected from H, Cl, F, —OC₂H₅,—OCH₃, and —OCH(CH₃)₂.
 7. A compound of claim 2 wherein R₂ is selectedfrom H, methyl, ethyl, methoxy, ethoxy, fluoromethyl, trifluoromethyl,aminomethyl and hydroxymethyl.
 8. A compound of claim 2 wherein R₃ isselected from H, —OH, methyl, methoxy, F and Cl.
 9. A compound of claim2 wherein R_(4a) is selected from —OH, methyl, ethyl, trifluoromethyl,methoxy, ethoxy, amino, F and Cl.
 10. A compound of claim 2 whereinR_(4b) is selected from methyl and F.
 11. A compound of claim 1 or claim2 selected from the group consisting of(S)-5-amino-N-(4-(3-aminopiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)-thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-methylpiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,5-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-(trifluoromethyl)piperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-ethylpiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluoro-4-hydroxyphenyl)thiazole-4-carboxamide,5-amino-N-(4-(3-amino-4-hydroxycyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-(3-amino-5-(fluoromethyl)piperidin-1-yl)-pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluoro-3-methoxyphenyl)thiazole-4-carboxamide,5-amino-2-(2,6-difluorophenyl)-N-(4-((1R,3S,5S)-3-hydroxy-5-methylcyclohexyl)pyridin-3-yl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-(3-amino-4-methylpiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(3-ethoxy-2,6-difluorophenyl)-thiazole-4-carboxamide,or a stereoisomer, tautomer, or pharmaceutically acceptable saltthereof.
 12. A compound of claim 1 or 2 selected from the groupconsisting of(S)-5-amino-N-(4-(3-aminopiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-methylpiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methyl-piperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,5-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-(trifluoromethyl)piperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((3S,5R)-3-amino-5-ethylpiperidin-1-yl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluoro-4-hydroxyphenyl)thiazole-4-carboxamide,5-amino-N-(4-(3-amino-4-hydroxycyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-(3-amino-5-(fluoromethyl)piperidin-1-yl)-pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluoro-3-methoxyphenyl)thiazole-4-carboxamide,5-amino-2-(2,6-difluorophenyl)-N-(4-((1R,3S,5S)-3-hydroxy-5-methylcyclohexyl)pyridin-3-yl)thiazole-4-carboxamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide,or a stereoisomer, tautomer, or pharmaceutically acceptable saltthereof.
 13. A method for treating a condition by modulation of ProvirusIntegration of Maloney Kinase (PIM kinase) activity comprisingadministering to a patient in need of such treatment an effective amountof a compound of any one of claims 1 through claim
 12. 14. A compositioncomprising a therapeutically effective amount of compound of any one ofclaims 1 through claim 12, or a stereoisomer, tautomer, orpharmaceutically acceptable salt thereof, together with apharmaceutically acceptable carrier.
 15. A method for inhibiting PIMkinase activity in a cell, comprising contacting the cell with aneffective amount of a compound of any one of claims 1 through claim 12.15. A method for treating a condition by modulation of ProvirusIntegration of Maloney Kinase (PIM kinase) activity comprisingadministering to a patient in need of such treatment an effective amountof a compound of any one of claims 1 through claim
 12. 16. A method forinhibiting PIM kinase activity in a patient, comprising administering tothe patient a composition comprising a pharmacologically effectiveamount of a compound of any one of claims 1 through claim
 12. 17. Amethod for treating a cancer disorder in a patient, comprisingadministering to the patient a composition comprising an amount of acompound of any one of claims 1 through claim 12 effective to inhibitPIM kinase activity in the patient.
 18. A compound of any one of claims1 through claim 12 for use as a therapeutic agent.
 19. A compound of anyone of claims 1 through claim 12 for use in manufacture of a medicamentfor the treatment of cancer.